Compositions relating to vitamin D

ABSTRACT

This invention relates to compositions, methods and kits for determining the presence and/or amount of vitamin D analytes, including vitamin D isomers, and metabolites thereof in a sample suspected of containing the same. Compounds include compounds of the Formula I, namely, (R1)p-(L)q-Z wherein R1, L, p and q are as defined herein. These compounds are capable of binding specifically to corresponding antibodies. Compounds and antibodies in accordance with the principles described herein can have therapeutic activity. These compounds may be administered in therapeutically effective amount, which is an amount to provide treatment of a particular disease state associated with vitamin D. Administration of the examples of compounds in accordance with the principles described herein can be by means of any of the accepted modes of administration for agents that serve similar utilities.

The subject application claims benefit under 35 USC § 119(e) of U.S.provisional Application No. 62/018,008, filed Jun. 27, 2014. The entirecontents of the above-referenced patent application are hereby expresslyincorporated herein by reference.

BACKGROUND

This invention relates to compositions, methods and kits for determiningthe presence and/or amount of vitamin D analytes, including vitamin Disomers, and metabolites thereof in a sample suspected of containing thesame.

The term “vitamin D” refers to a group of fat-soluble secosteroids. Inhumans, vitamin D is unique because it can be ingested ascholecalciferol (vitamin D₃) or ergocalciferol (vitamin D₂) and becausethe body can also synthesize it (from cholesterol) when sun exposure isadequate. Because of this latter property, vitamin D is considered bysome to be a non-essential dietary vitamin although most consider it anessential nutrient. Vitamin D has an important physiological role in thepositive regulation of calcium ion homeostasis. Vitamin D₃ is the formof the vitamin synthesized by animals. It is also a common supplementadded to milk products and certain food products as is vitamin D₂.

Both dietary and intrinsically synthesized vitamin D₃ must undergometabolic activation to generate bioactive metabolites. In humans, theinitial step of vitamin D₃ activation occurs primarily in the liver andinvolves hydroxylation to form the intermediate metabolite25-hydroxycholecalciferol. Calcidiol is the major form of Vitamin D₃ inthe circulatory system. Vitamin D₂ also undergoes similar metabolicactivation to 25-hydroxyvitamin D₂. Collectively these compounds arecalled 25-hydroxyvitamin D (abbreviated 25(OH)D) and they are the majormetabolites that are measured in serum to determine vitamin D status;25(OH)D and its epimers are both pre-hormones that need to be convertedinto 1,25(OH)D to exert biological functions. The comparison ofbioactivity of 1,25(OH)D versus that of 3-epi-1,25(OH)D is complex.

The vitamin D compounds 25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂are epimeric at the 3-position with the epimers being designated25-hydroxyvitamin D₃ and 3-epi-25-hydroxyvitamin D₃ and25-hydroxyvitamin D₂ and 3-epi-25-hydroxyvitamin D₂, respectively. Onlyone of the epimers of each of these epimeric compounds, namely,25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂, respectively, are active.The structures for the epimers of 25-hydroxyvitamin D₃ and25-hydroxyvitamin D₂ are set forth in FIG. 1.

Many small molecule compounds or haptens such as, for example, drugs andvitamins, exist in isomeric forms, of which only one form is active. Inorder to obtain an accurate measurement of the active form of ananalyte, the presence of the non-active isomer of the analyte must beaddressed. Measurements of both isomeric forms of an analyte, that is,active and non-active forms, can lead to inaccuracies that may bedetrimental to an individual depending on the function of the activeform of the analyte. Accurately assessing the level of each of a pair ofisomeric analytes in biological samples is important especially whereonly one of the isomers is active and measurements that include theamount of the non-active isomer distort the level of the analyte in asample. For example, measuring vitamin D levels in biological samples isimportant since vitamin D deficiency is related to a number of disordersin mammals. In infants, for example, vitamin D measurements that includeamounts of 3-epi isomers can lead to inaccurate assessment of vitamin Dlevels in the infant, which in turn can lead to a lack of propersupplementation. It is important to measure the active form of vitamin Dso that an infant can receive proper vitamin D therapy, if necessary.

Assessing vitamin D levels in biological samples is important sincevitamin D deficiency is related to a number of disorders in mammals.There is a need for reagents and methods for accurate and sensitivedeterminations of concentrations of vitamin D, epimeric forms of vitaminD, and vitamin D analogs and metabolites thereof in samples.

SUMMARY

Some examples in accordance with the principles described herein aredirected to a compound of the Formula I:(R¹)_(p)-(L)_(q)-Z, wherein

or H, wherein at least one R¹ is not H,

Y is O, S, CR, or NR⁴,

X is —O—(CH₂)_(n)—C(O)—, —(CH₂)_(w)—C(O)—,—(CH₂)_(w)—C(O)—(CH₂)_(x)—C(O)—, —(CH₂)_(w)—C(O)—NH(CH₂)_(y)—C(O)—,—NR³—C(O)—,

R is independently H or alkyl,

R² is independently H or alkyl,

R³, R⁴, R⁵ and R⁶ are independently H or alkyl, or R⁴ and R⁵ may betaken together to form a bond, or R⁵ and R⁶ may be taken together toform a bond,

R¹¹ is H, alkyl, or acyl,

n is an integer from 1 to 10,

w is an integer from 0 to 10,

x is an integer from 1 to 10,

y is an integer from 1 to 10,

p is an integer from 1 to 10,

L is a linking group,

q is 0 or 1, and

Z is OR⁸ wherein R⁸ is H, alkyl, or NR⁹R¹⁰ wherein R⁹ and R¹⁰ areindependently H or alkyl, a poly(amino acid) immunogenic carrier, anon-poly(amino acid) immunogenic carrier, a poly(amino acid) labelmoiety, a non-poly(amino acid) label moiety, a non-label poly(aminoacid) moiety, a non-immunogenic carrier poly(amino acid) moiety, or asupport; and including mixtures of two or more of the above compounds.

Some examples in accordance with the principles described herein aredirected to a compound of the Formula II:NHR^(1′)—(CH₂)_(r′)—NR^(1″)—((CH₂)_(r′)—NR^(1′″))_(s′)—(CH₂)_(r′)—NR^(7′)—Z′wherein

R^(1′), R^(1″) or R^(1′″) are each independently selected from

and H,

wherein at least one of R^(1′), R^(1″) or R^(1′″) is not H,

n′ is an integer from 1 to 10,

r′ is independently an integer from 1 to 10,

s′ is an integer from 1 to 10,

R^(7′) is H or alkyl,

R^(11′) is H, alkyl, or acyl, and

Z′ is a poly(amino acid) immunogenic carrier, a non-poly(amino acid)immunogenic carrier, a poly(amino acid) label moiety, a non-poly(aminoacid) label moiety, a non-label poly(amino acid) moiety, or anon-immunogenic carrier poly(amino acid) moiety; and including mixturesof two or more of the above compounds.

Some examples in accordance with the principles described herein aredirected to a compound of the Formula III:

wherein

W is O or N—O—(CH₂)_(n″)C(O)—Z″,

n″ is an integer from 1 to 10,

R^(11″) is H, alkyl, or acyl, and

Z″ is OR⁸ wherein R⁸ is H, alkyl, or NR⁹R¹⁰ wherein R⁹ and R¹⁰ areindependently H or alkyl; and including mixtures of two or more of theabove compounds.

Some examples in accordance with the principles described herein aredirected to methods of determining one or both of the presence and theamount of vitamin D in a sample suspected of containing vitamin D.

BRIEF DESCRIPTION OF DRAWINGS

The drawings provided herein are not to scale and are provided for thepurpose of facilitating the understanding of certain examples inaccordance with the principles described herein and are provided by wayof illustration and not limitation on the scope of the appended claims.

FIG. 1 is a depiction of the chemical formulas for the epimeric forms of25-hydroxyvitamin D₃ and 25-hydroxyvitamin D₂.

FIG. 2 is a schematic diagram of a synthesis of compounds in accordancewith examples in accordance with the principles described herein.

FIG. 3 is a schematic diagram of a synthesis of compounds in accordancewith examples in accordance with the principles described herein.

FIG. 4 is a schematic diagram of a synthesis of compounds in accordancewith examples in accordance with the principles described herein.

FIG. 5 is a schematic diagram of a synthesis of compounds in accordancewith examples in accordance with the principles described herein.

FIG. 6 is a schematic diagram of a synthesis of compounds in accordancewith examples in accordance with the principles described herein.

FIG. 7 is a schematic diagram of a synthesis of compounds in accordancewith examples in accordance with the principles described herein.

FIG. 8 is a schematic diagram of a synthesis of compounds in accordancewith examples in accordance with the principles described herein.

FIG. 9 illustrates a comparison of standard curves generated usingreagents with and without added anti-3-epimer-VD antibody.

FIG. 10 is a standard curve for an immunoassay for determination of3-epi-25(OH)D in samples using anti-3-epimer-VD antibody.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

Compounds

As mentioned above, some examples in accordance with the principlesdescribed herein are directed to compounds of the Formula I:(R¹)_(p)-(L)_(q)-Z wherein

or H, wherein at least one R¹ is not H,

Y is O, S, CR, or NR⁴,

X is —O—(CH₂)_(n)—C(O)—, —(CH₂)_(w)—C(O)—, or —NR³—C(O)—; in someexamples, X is —O—(CH₂)_(n)—C(O)— when Y is NR⁴, for example,—(CH₂)_(w)—C(O)—, —(CH₂)_(w)—C(O)—(CH₂)_(x)—C(O)—, or—(CH₂)_(w)—C(O)—NH(CH₂)_(y)—C(O)— when Y is O or S, for example, or—NR³—C(O)— when Y is CR, for example,

R is independently H or alkyl,

R² is independently H or alkyl,

R³, R⁴, R⁵ and R⁶ are independently H or alkyl, or R⁴ and R⁵ may betaken together to form a bond, or R⁵ and R⁶ may be taken together toform a bond,

R¹¹ is H, alkyl, or acyl,

n is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to6, or 1 to 5, or 1 to 4, or 1 to 3, or to 2, or 2 to 10, or 2 to 9, or 2to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10,or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9,or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8to 9, or 9 to 10, for example,

w is an integer from 0 to 10, 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7,or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3,or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5,or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to10, or 8 to 9, or 9 to 10, for example,

x is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to6, or 1 to 5, or 1 to 4, or 1 to 3, or to 2, or 2 to 10, or 2 to 9, or 2to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10,or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9,or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8to 9, or 9 to 10, for example,

y is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to6, or 1 to 5, or 1 to 4, or 1 to 3, or to 2, or 2 to 10, or 2 to 9, or 2to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10,or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9,or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8to 9, or 9 to 10, for example,

p is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to6, or 1 to 5, or 1 to 4, or 1 to 3, or to 2, or 2 to 10, or 2 to 9, or 2to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10,or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9,or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8to 9, or 9 to 10, for example,

L is a linking group,

q is 0 or 1, and

Z is OR⁸ wherein R⁸ is H, alkyl, or NR⁹R¹⁰ wherein R⁹ and R¹⁰ areindependently H or alkyl, a poly(amino acid) immunogenic carrier moiety,a non-poly(amino acid) immunogenic carrier moiety, a poly(amino acid)label moiety, a non-poly(amino acid) label moiety, a non-labelpoly(amino acid) moiety, a non-immunogenic carrier poly(amino acid)moiety, or a support; and including mixtures of two or more of the abovecompounds.

As used herein, the term “alkyl” includes those alkyl groups of adesignated number of carbon atoms of either a straight, branched, orcyclic configuration. Examples of “alkyl” include, but are not limitedto, methyl, ethyl, propyl, isopropyl, butyl, sec- and tert-butyl,pentyl, hexyl, heptyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, norbornyl, for example. In some examples, alkyl contains 1to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to 6, or 1 to 5, or 1 to 4,or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or 2 to 8, or 2 to 7, or 2to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10, or 3 to 9, or 3 to 8,or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4 to 10, or 4 to 9, or 4to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to 10, or 5 to 9, or 5 to 8,or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9, or 6 to 8, or 6 to 7, or 7to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8 to 9, or 9 to 10, carbonatoms, which may be unsubstituted or one or more of which may besubstituted by one or more of hydroxy, alkoxy of 1 to 5, or 1 to 4, of 1to 3, or 1 to 2, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 5, or 3 to 4,or 4 to 5 carbon atoms.

As used herein, the term “acyl” means R¹²C(O)— where R¹² is alkyl oraryl.

As used herein, the term “aryl” means an organic radical derived from anaromatic hydrocarbon by the removal of one atom and containing one ormore aromatic rings, usually one to four aromatic rings, such as, e.g.,phenyl (from benzene), naphthyl (from naphthalene), etc., e.g., phenyl,naphthyl, phenanthryl.

As used herein, the phrase “linking group” refers to a chemical moietythat may comprise about 2 to about 50 atoms, or 4 to about 30 atoms, notcounting hydrogen and may comprise a chain of from 2 to about 30 atoms,or 3 to about 20 atoms, each independently selected from the groupnormally consisting of carbon, oxygen, sulfur, nitrogen, andphosphorous. In some examples, part or all of the linking group may be aportion of the molecule being linked such as, but not limited to, anamino acid residue on a poly(amino acid), for example. The number ofheteroatoms in the linking group may be in the range from 0 to about 20,or 1 to about 15, or about 2 to about 10. The linking group may bealiphatic or aromatic. When heteroatoms are present, oxygen is normallypresent as oxo or oxy, bonded to carbon, sulfur, nitrogen orphosphorous, nitrogen is normally present as nitro, nitroso or amino,normally bonded to carbon, oxygen, sulfur or phosphorous; sulfur isanalogous to oxygen; while phosphorous is bonded to carbon, sulfur,oxygen or nitrogen, usually as phosphonate and phosphate mono- ordiester. Common functionalities in forming a covalent bond between thelinking group and the molecule to be conjugated are alkylamine, amidine,thioamide, ether, urea, thiourea, guanidine, azo, thioether andcarboxylate, sulfonate, and phosphate esters, amides and thioesters.

For the most part, when a linking group has a linking functionality(functionality for reaction with a moiety) such as, for example, anon-oxocarbonyl group including nitrogen and sulfur analogs, a phosphategroup, an amino group, alkylating agent such as halo or tosylalkyl, oxy(hydroxyl or the sulfur analog, mercapto) oxocarbonyl (e.g., aldehyde orketone), or active olefin such as a vinyl sulfone or α-, β-unsaturatedester, these functionalities are linked to amine groups, carboxylgroups, active olefins, alkylating agents, e.g., bromoacetyl. Where anamine and carboxylic acid or its nitrogen derivative or phosphoric acidare linked, amides, amidines and phosphoramides are formed. Wheremercaptan and activated olefin are linked, thioethers are formed. Wherea mercaptan and an alkylating agent are linked, thioethers are formed.Where aldehyde and an amine are linked under reducing conditions, analkylamine is formed. Where a ketone or aldehyde and a hydroxylamine(including derivatives thereof where a substituent is in place of thehydrogen of the hydroxyl group) are linked, an oxime functionality(═N—O—) is formed. Where a carboxylic acid or phosphate acid and analcohol are linked, esters are formed.

As used herein, the term “immunogenic carrier” means a group or moietythat is conjugated to a hapten. The conjugate of the immunogenic carrierand the hapten may be injected into an organism capable of eliciting animmune response such as, but not limited to, a mammal, an avian (e.g.,chicken or pigeon), an amphibian, or a reptile; or the conjugate may beused to inoculate an in vitro sample (mammalian, including human, avian,amphibian or reptile) or otherwise may be employed in a technique toproduce a binding partner for the hapten.

The phrase “binding partner” refers to a molecule that is a member of aspecific binding pair, which is one of two different molecules thatspecifically binds to and is thereby defined as complementary with theother molecule. For example, one member of the specific binding pair mayhave an area on the surface or in a cavity that specifically binds to aparticular spatial and polar organization of the other member of thespecific binding pair. The binding partner may be, by way ofillustration and not limitation, an antibody or an aptamer (e.g.,nucleic acid aptamer or peptide aptamer), for example. In one example,an immunogenic carrier may be employed as an immunogen to induce animmune response and elicit production of a binding partner for a hapten.Other techniques include phage display and in vitro selection.Immunogenic carriers are also sometimes referred to as antigeniccarriers. In some examples in accordance with the principles describedherein, immunogens comprising immunogenic carriers, including poly(aminoacid) and non-poly(amino acid) immunogenic carriers, are synthesized andused to prepare antibodies. Haptens are compounds capable of bindingspecifically to corresponding antibodies, but do not themselves act asimmunogens (or antigens) for preparation of the antibodies.Consequently, a hapten is linked to an immunogenic carrier, which may beemployed, for example, to raise antibodies.

The molecular weight range (in Daltons) for poly(amino acids) that areimmunogenic carriers is about 5,000 to about 10,000,000, or about 20,000to about 600,000, or about 25,000 to about 250,000, for example.“Poly(amino acid) immunogenic carrier moieties” include proteins suchas, for example, albumins, serum proteins, e.g., globulins, ocular lensproteins and lipoproteins. Illustrative proteins include, but are notlimited to, bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH),egg ovalbumin, and bovine gamma-globulin (BGG), for example.“Non-poly(amino acid) immunogenic carrier moieties” includepolysaccharides, nucleic acids and particles (biologic and syntheticmaterials). A wide variety of immunogenic carriers are disclosed inDavalian, et al., U.S. Pat. No. 5,089,390, column 4, line 57 to column5, line 5, which is incorporated herein by reference.

As mentioned above, the immunogenic carrier moiety may be apolysaccharide, which is a high molecular weight polymer ofmonosaccharides that may be prepared naturally or synthetically andusually involves repeated condensations of monosaccharides. Examples ofpolysaccharides are starches, glycogen, cellulose, carbohydrate gums,such as gum arabic, agar, and so forth. The polysaccharide can alsocontain poly(amino acid) residues and/or lipid residues.

As used herein, the term “label” includes poly(amino acid) labels andnon-poly(amino acid) labels. The term “poly(amino acid) label moieties”includes labels that are proteins such as, but not limited to, enzymes,antibodies, peptides, and immunogens, for example. With label proteinssuch as, for example, enzymes, the weight average molecular weight rangewill be from about 10,000 to about 600,000 or from about 10,000 to about300,000. There is usually at least one compound in accordance with theprinciples described herein (analog group) per about 200,000 molecularweight, or at least about 1 per about 150,000 molecular weight, or atleast about 1 per about 100,000 molecular weight, or at least about 1per about 50,000 molecular weight, or at least about 1 per 40,000,molecular weight, or at least about 1 per 30,000 molecular weight, or atleast 1 per 20,000 molecular weight, or at least one per 10,000molecular, or at least one per 5,000 molecular weight, for example, ofthe protein. In the case of enzymes, the number of analog groups isusually from 1 to about 20, about 2 to about 15, about 3 to about 12, orabout 6 to about 10.

Enzymes include, by way of illustration and not limitation, redoxenzymes such as, for example, dehydrogenases, e.g., glucose-6-phosphatedehydrogenase (G6PDH) and lactate dehydrogenase; enzymes that involvethe production of hydrogen peroxide and the use of the hydrogen peroxideto oxidize a dye precursor to a dye such as, for example, horseradishperoxidase, lactoperoxidase and microperoxidase; hydrolases such as, forexample, alkaline phosphatase and β-galactosidase; luciferases such as,for example firefly luciferase, and bacterial luciferase; transferases;combinations of enzymes such as, but not limited to, saccharideoxidases, e.g., glucose and galactose oxidase, or heterocyclic oxidases,such as uricase and xanthine oxidase, coupled with an enzyme thatemploys hydrogen peroxide to oxidize a dye precursor, that is, aperoxidase such as horseradish peroxidase, lactoperoxidase ormicroperoxidase, for example.

As used herein, the term “non-poly(amino acid) labels” includes thoselabels that are not proteins. The non-poly(amino acid) label is capableof being detected directly or is detectable through a reaction thatproduces a detectable signal. The non-poly(amino acid) label can beisotopic or non-isotopic and can be, by way of illustration and notlimitation, a radioisotope, a luminescent compound (which includes, butis not limited to fluorescent compounds and chemiluminescent compounds,for example), a polynucleotide coding for a catalyst, a promoter, a dye,a coenzyme, an enzyme substrate, a radioactive group, a small organicmolecule (molecular weight 200 to 2,000), a particle, and an amplifiablepolynucleotide sequence, for example.

As mentioned above, a “small organic molecule” has a molecular weight ofabout 200 to about 2,000, or about 200 to about 1,500, or about 200 toabout 1,000, or about 200 to about 500. Such “small organic molecules”include, but are not limited to, biotin, fluorescent molecules (such asfluorescein and rhodamine, for example), chemiluminescent molecules, anddinitrophenol, for example. A binding partner for a small organicmolecule is a molecule that specifically recognizes and binds to thesmall molecule. Binding partners for a small molecule are defined by thenature of the small molecule and include, but are not limited to,avidin, streptavidin, antibody for the small organic molecule (whichinclude, but are not limited to, antibody for a fluorescent molecule(such as antibody for fluorescein and antibody for rhodamine, forexample), antibody for a chemiluminescent molecule, and antibody fordinitrophenol, for example.

As used herein, the terms “non-label poly(amino acid) moiety” and“non-immunogenic carrier poly(amino acid) moiety” refer to poly(aminoacids) that are not normally considered labels or immunogenic carriersalthough such moieties may be labels or immunogenic carriers in certaincircumstances. For example, an antibody may not be considered a labelbut may be a label if the antibody is modified to include a signalproducing moiety or part of a signal producing system. Furthermore, anantibody may not be considered as an immunogenic carrier but isnonetheless capable of being an immunogenic carrier in certaincircumstances because of it higher molecular weight.

In some examples the non-poly(amino acid) label may be selected from thegroup consisting of supports, magnetic particles, acridinium esters, acombination of magnetic particles and acridinium esters (such as, forexample, acridinium ester labeled paramagnetic particles),chemiluminescent particles and sensitizer particles.

The term “covalent” refers to attachment of molecules such as by adirect connection, e.g., a chemical bond between the molecules. The term“non-covalent” refers to attachment of molecules involving specificbinding between complementary specific binding pair (sbp) members thatare attached to the molecules.

In some examples compounds in accordance with the principles describedherein may be associated with a support, for example, by covalent ornon-covalent binding. As mentioned above, in some examples in accordancewith the principles described herein, Z may be a support, which may becomprised of an organic or inorganic, solid or fluid, water insolublematerial and which may be transparent or partially transparent. Thesupport can have any of a number of shapes, such as, but not limited to,a particle (particulate support) including bead, a film, a membrane, atube, a well, a strip, a rod, a fiber, or a planar surface such as,e.g., a plate or paper, for example. The support may or may not besuspendable in the medium in which it is employed. Examples ofsuspendable supports are polymeric materials such as latex, lipidbilayers or liposomes, oil droplets, cells and hydrogels, and magneticparticles, for example. Other support compositions include polymers,such as, by way of illustration and not limitation, nitrocellulose,cellulose acetate, poly (vinyl chloride), polyacrylamide, polyacrylate,polyethylene, polypropylene, poly(4 methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), for example, either used by themselves or in conjunction withother materials. The support may or may not be further labeled with adye, catalyst or other detectable group, for example.

In some examples in accordance with the principles described herein, thesupport may be a particle. The particles have an average diameter of atleast about 0.02 microns and not more than about 100 microns. In someexamples, the particles have an average diameter from about 0.05 micronsto about 20 microns, or from about 0.3 microns to about 10 microns. Theparticle may be organic or inorganic, swellable or non-swellable, porousor non-porous, preferably of a density approximating water, generallyfrom about 0.7 g/mL to about 1.5 g/mL, and composed of material that canbe transparent, partially transparent, or opaque. The particles can bebiological materials such as cells and microorganisms, e.g.,erythrocytes, leukocytes, lymphocytes, hybridomas, streptococcus,Staphylococcus aureus, and E. coli, viruses, for example. The particlescan also be particles comprised of organic and inorganic polymers,liposomes, latex particles, magnetic or non-magnetic particles,phospholipid vesicles, chylomicrons, lipoproteins, and the like. In someexamples, the particles are chromium dioxide (chrome) particles or latexparticles.

Magnetic particles include paramagnetic particles, ferromagneticparticles and diamagnetic particles. Such particles include, but are notlimited to, transition metals of periods 4-7 of the Periodic Tableincluding chromium, copper, cobalt, aluminum, manganese, iron, andnickel, for example.

Chemiluminescent particles are particles that have associated therewitha chemiluminescent compound. The phrase “associated therewith” as usedherein means that a compound such as, for example, a chemiluminescentcompound and a particle may be associated by direct or indirect bonding,adsorption, absorption, incorporation, or solution, for example.Examples of chemiluminescent compounds that may be utilized are thoseset forth in U.S. Pat. Nos. 5,340,716 and 6,251,581, the relevantdisclosures of which are incorporated herein by reference. In someexamples in accordance with the principles described herein, thechemiluminescent compound is a photoactivatable substance that undergoesa chemical reaction upon direct or sensitized excitation by light orupon reaction with singlet oxygen to form a metastable reaction productthat is capable of decomposition with the simultaneous or subsequentemission of light, usually within the wavelength range of 250 to 1200nm. The term “photoactivatable” includes “photochemically activatable”.In some examples, the chemiluminescent compounds are those that reactwith singlet oxygen to form dioxetanes or dioxetanones. The latter areusually electron rich olefins. Exemplary of such electron rich olefinsare enol ethers, enamines, 9-alkylidene-N-alkylacridans,arylvinylethers, dioxenes, arylimidazoles, 9-alkylidene-xanthanes andlucigenin. Other compounds include luminol and other phthalhydrazidesand chemiluminescent compounds that are protected from undergoing achemiluminescent reaction by virtue of their being protected by aphotochemically labile protecting group, such compounds including, forexample, firefly luciferin, aquaphorin, and luminol. Examples of suchchemiluminescent compounds that may be utilized are those set forth inU.S. Pat. No. 5,709,994, the relevant disclosure of which isincorporated herein by reference.

Sensitizer particles are particles that have associated therewith asensitizer compound, which includes, but is not limited to, aphotosensitizer compound. Examples of sensitizer compounds that may beutilized are those set forth in U.S. Pat. Nos. 5,340,716 and 6,251,581,the relevant disclosures of which are incorporated herein by reference.

A photosensitizer is a sensitizer for generation of singlet oxygenusually by excitation with light. In some examples, the photosensitizerabsorbs at a longer wavelength than the chemiluminescent compound andhas a lower energy triplet than the chemiluminescent compound. Thephotosensitizer can be photoactivatable (e.g., dyes and aromaticcompounds). The photosensitizer is usually a compound comprised ofcovalently bonded atoms, usually with multiple conjugated double ortriple bonds. The compound should absorb light in the wavelength rangeof 200-1100 nm, usually 300-1000 nm, preferably 450-950 nm. Typicalphotosensitizers include, but are not limited to, acetone, benzophenone,9-thioxanthone, eosin, 9,10-dibromoanthracene, methylene blue,metallo-porphyrins (e.g., hematoporphyrin), phthalocyanines,chlorophylls, rose bengal, buckminsterfullerene, for example, andderivatives of these compounds. Examples of other photosensitizers areenumerated in N. J. Turro, “Molecular Photochemistry”, page 132, W. A.Benjamin Inc., N.Y. 1965. The photosensitizer assists photoactivationwhere activation is by singlet oxygen. Usually, the photosensitizerabsorbs light and the thus formed excited photosensitizer activatesoxygen to produce singlet oxygen, which reacts with the chemiluminescentcompound to give a metastable luminescent intermediate.

In the formulas set forth herein, a squiggle line through a bondindicates the point of attachment of a moiety in the formula.

Some examples in accordance with the principles described herein aredirected to Formula IV compounds, which are compounds of Formula Iwherein:

or H, wherein at least one R¹ is not H; and including mixtures of two ormore of the above compounds.

Some examples in accordance with the principles described herein aredirected to derivatives of(7aS,E)-4-((Z)-2-((R)-5-hydroxy-2-methylene-cyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-one(HMCHEMOIO derivatives), or Formula V compounds, which are compounds ofFormula I wherein:

or H, wherein at least one R¹ is not H; and including mixtures of two ormore of the above compounds.

Some examples in accordance with the principles described herein aredirected to Formula VI compounds, which are compounds of Formula Iwherein(R¹)_(p)-(L)_(q)- isNHR¹—(CH₂)_(r)—NR¹—((CH₂)_(r)—NR¹)_(s)—(CH₂)_(r)—NR⁷— wherein R¹ isindependently

or H,

wherein at least one R¹ is not H,

p, q and n are as defined above,

r is independently an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10,or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5,or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8,or 8 to 10, or 8 to 9, or 9 to 10, for example,

s is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9,or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8to 9, or 9 to 10, for example, and

R⁷ is H or alkyl; and including mixtures of two or more of the abovecompounds.

Some examples in accordance with the principles described herein aredirected to compounds of Formula II:NHR^(1′)—(CH₂)_(r′)—NR^(1″)—((CH₂)_(r′)—NR^(1′″))_(s′)—(CH₂)_(r′)—NR^(7′)—Z′whereinR^(1′), R^(1″) or R^(1′″) are each independently selected from the groupconsisting of

and H,

wherein at least one of R^(1′), R^(1″) or R^(1′″) is not H,

n and w are as defined above,

r′ is independently an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1to 7, or 1 to 6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10,or 2 to 9, or 2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2to 3, or 3 to 10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5,or 3 to 4, or 4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4to 5, or 5 to 10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to10, or 6 to 9, or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8,or 8 to 10, or 8 to 9, or 9 to 10, for example,

s′ is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to6, or 1 to 5, or 1 to 4, or 1 to 3, or to 2, or 2 to 10, or 2 to 9, or 2to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to 10,or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or 4to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9,or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8to 9, or 9 to 10, for example,

R^(7′) is H or alkyl,

R^(11′) is H, alkyl, or acyl, and

Z′ is a poly(amino acid) immunogenic carrier, a non-poly(amino acid)immunogenic carrier, a poly(amino acid) label moiety, a non-poly(aminoacid) label moiety, a non-label poly(amino acid) moiety, anon-immunogenic carrier poly(amino acid) moiety, or a support; andincluding mixtures of two or more of the above compounds.

In some examples s′ is 1. In some examples R^(7′) is H. In some examplesr′ is 2. In some examples R^(1″) and R^(1′″) are H; in some examples R1′and R^(1′″) are H; and in some examples R^(1′) and R^(1″) are H. In someexamples none of R^(1′), R^(1″) and R^(1′″) is H, that is, R^(1′),R^(1″) and R^(1′″) are all

Some examples in accordance with the principles described herein aredirected to compounds of Formula III:

wherein

W is O or N—O—(CH₂)_(n)C(O)—Z″,

n is an integer from 1 to 10, or 1 to 9, or 1 to 8, or 1 to 7, or 1 to6, or 1 to 5, or 1 to 4, or 1 to 3, or 1 to 2, or 2 to 10, or 2 to 9, or2 to 8, or 2 to 7, or 2 to 6, or 2 to 5, or 2 to 4, or 2 to 3, or 3 to10, or 3 to 9, or 3 to 8, or 3 to 7, or 3 to 6, or 3 to 5, or 3 to 4, or4 to 10, or 4 to 9, or 4 to 8, or 4 to 7, or 4 to 6, or 4 to 5, or 5 to10, or 5 to 9, or 5 to 8, or 5 to 7, or 5 to 6, or 6 to 10, or 6 to 9,or 6 to 8, or 6 to 7, or 7 to 10, or 7 to 9, or 7 to 8, or 8 to 10, or 8to 9, or 9 to 10, for example,

R^(11″) is H, alkyl, or acyl, and

Z″ is OR⁸ wherein R⁸ is H, alkyl, or NR⁹R¹⁰ wherein R⁹ and R¹⁰ areindependently H or alkyl; and including mixtures of two or more of theabove compounds.

Some examples in accordance with the principles described herein aredirected to compounds of the Formula VII:

Some examples in accordance with the principles described herein aredirected to pharmaceutical compositions comprising an effective amountof one or more of the compounds identified above such as, for example,compounds of the Formula III and compounds of the Formula VII, andincluding mixtures of two or more of the above compounds.

Preparation of Compounds

Examples of methods of preparing compounds that are HMCHEMOIOderivatives in accordance with the principles described herein arediscussed below, by way of illustration and not limitation. Otherapproaches may be employed to form the above compounds and othercompounds consistent with the principles described herein.

An example of a preparation of a compound of Formula VII((7aS,E)-4-((Z)-2-((R)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-one)is set forth in FIG. 2. Referring to FIG. 2, compound of the FormulaVIII, wherein R¹¹O is acetyl, is treated to form ketal of Formula IX. Inone example, compound of the Formula VIII is treated with ethyleneglycol in an aromatic solvent such as benzene in the presence of astrong organic acid such as, for example, p-toluene sulfonic acid, underconditions (temperature and time) for forming a ketal. In some examplesthe reaction is conducted at reflux for a period of about 10 hours toabout 24 hours, or about 12 to about 20 hours.

Compound of the Formula IX is treated to introduce a halide group suchas, for example, a chloride group or a bromide group. In one example,compound of Formula IX is treated with N-bromosuccinimide and a freeradical initiator such as, for example, azobisisobutyronitrile in anorganic solvent such as, for example, an alkane (e.g., hexane), forexample, under conditions for introducing a bromide group into thecompound of Formula IX to give a compound of the Formula X. In someexamples the reaction is conducted at reflux for about 30 minutes.

The halide of the compound of Formula X is removed to give a compound ofthe Formula XI having two double bonds that are conjugated. In oneexample, the compound of Formula X is treated with mild base such as,for example, tetra-n-butylammonium fluoride, in a polar organic solventsuch as, for example, an ether (e.g., tetrahydrofuran), for example,under conditions for removing a hydrogen halide to form a double bond(compound of Formula XI). In some examples the temperature during thereaction is about 15° C. to about 25° C. The time period of the reactionis about 2 hours.

The acetyl group (R¹¹) of the compound of Formula XI is removed bytreatment with an inorganic base such as, for example, sodium hydroxideor potassium hydroxide, in a polar organic solvent such as, for example,an alkanol (e.g., methanol or ethanol). The reaction components aresubjected to conditions for removing the acetyl group to give compoundof the Formula XII. In some examples the temperature during the reactionis about 15° C. to about 25° C., or at room temperature. The time periodof the reaction is about 4 hours to about 8 hours.

The ketal group of the compound of Formula XII is removed, for example,using a strong organic acid such as, for example, p-toluene sulfonicacid, in a mixture of water and a polar organic solvent such as, forexample, acetone, under conditions for removing a ketal to yield thecompound of Formula XIII. In some examples the temperature during thereaction is about 15° C. to about 25° C. The time period of the reactionis about 12 hours to about 48 hours.

The compound of Formula VII is formed by treating the compound ofFormula XIII with reagents for opening a ring such as, for example,treatment with UV light (photo reaction) in a polar organic solvent suchas, for example, an alkanol (e.g., methanol or ethanol), an ether (e.g.,ethyl ether), a ketone (e.g., acetone), or a combination of two or moreof the above, under conditions for opening a ring of Formula XIII. Insome examples the temperature during the photo reaction is about −20° C.to about 0° C. The time period of the photo reaction is about 1 to about10 minutes, or about 3 to about 5 minutes. The photo reaction isfollowed by refluxing the intermediate in ethanol for about 3 hours.

FIG. 3 depicts, by way of illustration and not limitation, an example ofa method of preparing a compound of the Formula IIa (where Z is BSA, byway of example and not limitation). Referring to FIG. 3, compound ofFormula VII is reacted with aminooxyacetic acid (XIX) to form oxime ofthe Formula XX(2-((7aS,E)-4-((Z)-2-((R)-5-hydroxy-2-methylenecyclohexylidene)ethylidene)-7a-methyloctahydro-1H-inden-1-ylideneaminooxy)aceticacid). The reaction is carried out in an organic solvent such as, forexample, an alcohol (e.g., methanol or ethanol), under conditions forforming an oxime. In some examples the temperature during the reactionis about 10° C. to about 30° C., or about 15° C. to about 25° C. Thetime period of the reaction is about 1 hour to about 30 hours, or about2 hours to about 24 hours.

In a separate reaction, a poly(amino)acid immunogenic carrier (in thisexample, BSA (Z′ of Formula II is BSA), by way of illustration and notlimitation) is combined with a compound of the formula XXI to form acompound of the formula XXIa. The reaction is carried out in an aqueousbuffered medium at a pH of about 5.0 to about 7.0, or about 5.5 to about6.5, or about 6. An activation agent for facilitating the reaction ofthe carboxylic acid functionality of BSA with one or more of the aminegroup(s) of XXI is included in the reaction medium. Such coupling agentsinclude, but are not limited to,1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC),N-hydroxysuccinimide (NHS), orN,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate, orcombinations of two or more of the above. The reaction is carried outunder conditions for forming an amide. In some examples the temperatureduring the reaction is about 15° C. to about 25° C. The time period ofthe reaction is about 3 hours to about 24 hours, or about 4 hours toabout 20 hours, or about 4 hours to about 10 hours, for example.

The compound of the Formula XX is treated with an activation agent suchas, but are not limited to,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC),N-hydroxysuccinimide (NHS), orN,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroborate, orcombinations of two or more of the above, for example, to form activatedXX. The reaction is carried out in a polar organic solvent such as, forexample, an alkanol (e.g., methanol or ethanol), an ether (e.g., ethylether or tetrahydrofuran), a ketone (e.g., acetone), dimethylsulfoxide,acetonitrile, dichloromethane, or dimethylformamide (DMF), or acombination of two or more of the above, which may also contain water.In some examples the temperature during the reaction is about 15° C. toabout 30° C., or about 20° C. to about 25° C., or about roomtemperature. The time period of the reaction is about 12 hours to about36 hours, or about 20 to about 30 hours. The activated XX is reactedwith the compound of the Formula XXIa to give a compound of the FormulaIIa. As shown, R^(1′) is the moiety of compound of the Formula IIa andR^(1″) and R^(1′″) are each H. However, consistent with the principlesdescribed herein, the resulting product may be a mixture of compoundswherein in one compound of the mixture R^(1′) is the moiety of thecompound of the Formula IIa, and in another compound of the mixtureR^(1′) and R^(1″) are both the moiety of compound of the Formula IIa,and in another compound of the mixture R^(1′) and R^(1′″) are both themoiety of compound of the Formula IIa, and in another compound of themixture all three of R^(1′) and R^(1″) and R^(1′″) are the moiety ofcompound of the Formula IIa. The reaction is carried out in an organicsolvent such as, for example, DMF or DMSO, for example, under conditionsfor forming an amide. In some examples the temperature during thereaction is about 15° C. to about 30° C., or about 20° C. to about 25°C., or about room temperature. The time period of the reaction is about12 hours to about 36 hours, or about 20 to about 30 hours.

FIG. 4 depicts, by way of illustration and not limitation, an alternateexample of a method of preparing a compound of the Formula VIa andconversion of the compound of Formula VIa to a compound of the FormulaIIa (where Z is BSA, by way of example and not limitation). Referring toFIG. 4, compound of Formula VII is reacted with aminooxyacetic acid(XIX) to form oxime of the Formula XX. The reaction is carried out in anorganic solvent such as, for example, an alcohol (e.g., methanol orethanol), under conditions for forming an oxime. In some examples thetemperature during the reaction is about 10° C. to about 30° C., orabout 15° C. to about 25° C. The time period of the reaction is about 1hour to about 30 hours, or about 2 hours to about 24 hours.

Oxime of the Formula XX is combined with a compound of the formula XXIto form compound of the formula VIa. The reaction is carried out in anorganic solvent such as, for example, an alkane (e.g., hexane orpentane, under conditions for forming an amide. In some examples thetemperature during the reaction is about 15° C. to about 25° C. The timeperiod of the reaction is about 10 hours to about 48 hours.

The compound of Formula VIa is reacted with a poly(amino)acidimmunogenic carrier (Z′ of Formula II is BSA, by way of illustration andnot limitation) to give a compound of the Formula IIa. As shown, R^(1′)is the moiety of compound of the Formula IIa and R^(1″) and R^(1′″) areeach H. However, consistent with the principles described herein, theresulting product may be a mixture of compounds wherein in one compoundof the mixture R^(1′) is the moiety of the compound of the Formula IIa,and in another compound of the mixture R^(1′) and R^(1″) are both themoiety of compound of the Formula IIa, and in another compound of themixture R^(1′) and R^(1′″) are both the moiety of compound of theFormula IIa, and in another compound of the mixture all three of R^(1′)and R^(1″) and R^(1′″) are the moiety of compound of the Formula IIa.The reaction is carried out in an organic solvent such as, for example,an alkane (e.g., hexane or pentane), under conditions for forming anamide. In some examples the temperature during the reaction is about 15°C. to about 25° C. The time period of the reaction is about 10 hours toabout 48 hours.

Another example of a method of preparing examples of compounds inaccordance with the principles described herein is described, by way ofillustration and not limitation, with reference to FIG. 5. Otherapproaches may be employed to form the compounds consistent with theprinciples described herein. The preparation of a compound of FormulaXVIII (Y is C in Formula I) is set forth in FIG. 5. Referring to FIG. 5,compound of the Formula VII (prepared, for example, as discussed in FIG.3) is treated to protect the free hydroxyl group (R¹³ is a protectinggroup) to form a compound of the Formula XIV. The conditions of thereaction are dependent on the nature of the protecting group, forexample.

In one example, compound of the Formula VII is treated withtert-butyldimethylsilyl chloride under conditions for forming a silylether. The reaction is carried out in a polar organic solvent such as,for example, pyridine, dimethylsulfoxide, an ether (e.g.,tetrahydrofuran, ethyl ether or 1,4-dioxane), acetonitrile,dichloromethane, or dimethylformamide (DMF). In some examples thetemperature during the reaction is about 10° C. to about 30° C., orabout 15° C. to about 25° C. The time period of the reaction is about 1hour to about 30 hours, or about 2 hours to about 24 hours.

Examples of protecting groups, by way of example and not limitation, aresilyl groups (such as, but not limited to, trimethylsilyl,trimethylsilyl, tert-butyldimethylsilyl, tri-isopropylsilyl,tert-butyldiphenylsilyl, for example), t-butoxycarbonyl (t-Boc),fluorenylmethyloxycarbonyl (Fmoc), acetaminomethyl (Acm), triphenylmethyl (Trt), benzyloxycarbonyl, biphenylisopropyloxycarbonyl,1-amyloxycarbonyl, isobornyloxycarbonyl,alpha-dimethyl-3,5-dimethoxybenxyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-1,1-dimentyl-ethoxycarbonyl, bromobenzyloxy, carbamyl, andformyl, for example.

A compound of the Formula XV is formed from the compound of Formula XIVby, for example, reaction with an alpha-haloester in the presence ofzinc (Reformatsky reaction). In this example, the compound of FormulaXIV is treated with a Reformatsky reagent such as, for example,BrZnCH₂COOR¹⁴ (Zn and ethyl alpha-bromoacetate in the example shown,w=1) to give the compound of the Formula XV. The reaction is carried outin an organic solvent such as, for example, an ether (e.g.,tetrahydrofuran or ethyl ether) or an aromatic solvent (e.g., benzene ortoluene). In some examples the temperature during the reaction is about4° C. to about 100° C., or about 10° C. to about 90° C. The time periodof the reaction is about 4 hours to about 24 hours.

The free hydroxyl group of the compound of Formula XV is reduced to forma compound of the Formula XVI. The compound of Formula XV may be treatedto reduce the hydroxy group by methods that include, but are not limitedto, tosylate ester formation followed by treatment with a metal hydridesuch as, for example, LiAlH₄ or NaBH₄; removal of the hydroxyl to forman alkene (such as, for example, by treatment with a concentratedorganic acid, e.g., concentrated sulfuric acid or concentratedhydrochloric acid) followed by hydrogenation in the presence of acatalyst such as, for example, platinum or palladium; for example. Theconditions for the reaction depend on one or both of the nature of thereagents employed and the nature of the solvent, for example.

The resulting compound of the Formula XVI is treated to hydrolyze theester group to a carboxylic acid group (de-esterification reaction) togive the compound of the Formula XVII. Numerous approaches are availablefor de-esterification and include, but are not limited to,saponification or treatment with an aqueous base such as, for example,NaOH or KOH with heat; or acid hydrolysis or treatment with an aqueousacid such as, for example, an aqueous mineral acid (such as, forexample, hydrochloric acid or sulfuric acid), for example. Theconditions for the reaction are dependent on one or more of the natureof the reagents and the nature of the ester, for example.

A compound of the Formula XVIII is formed by removal of protecting groupR¹³ from the compound of Formula XVII. Various approaches may beemployed for removal of protecting groups and include, but are notlimited to, treatment with dilute mineral acid (such as, for example,hydrochloric acid or sulfuric acid; treatment with an organic acid (suchas, for example, acetic acid, in a polar organic solvent (such as, forexample, an alkanol (e.g., methanol or ethanol), an ether (e.g., ethylether or tetrahydrofuran), a ketone (e.g., acetone), dimethylsulfoxide,acetonitrile, dichloromethane, or dimethylformamide (DMF), or acombination of two or more of the above, which may also contain water,under conditions for removing the protecting group to yield the compoundof Formula XVIII. The conditions for the reaction are dependent on oneor more of the nature of the reagents and the nature of the protectinggroup, for example.

FIG. 6 depicts, by way of illustration and not limitation, an example ofa method of preparing a compound of the Formula VIb and conversion ofthe compound of Formula VIb to a compound of the Formula IIb (where Z′is KLH, by way of example and not limitation). Referring to FIG. 6,compound of Formula XVIII is treated to activate the carboxylic acidgroup such as, but not limited to, reaction to form anN-hydroxysuccinimide (NHS) ester (compound of the Formula XXII). Thereaction is carried out in a polar organic solvent such as, for example,dimethylsulfoxide, an ether (e.g., tetrahydrofuran or ethyl ether),acetonitrile, dichloromethane, or dimethylformamide (DMF), for example,under conditions for forming an NHS ester. In some examples thetemperature during the reaction is about 15° C. to about 25° C. The timeperiod of the reaction is about 4 hours to about 48 hours.

NHS ester of the Formula XXII is combined with a compound of the formulaXXI to form compound of the formula VIb. The reaction is carried out inan organic solvent such as, for example, an alkane (e.g., hexane orpentane), for example, under conditions for forming an amide. In someexamples the temperature during the reaction is about 15° C. to about25° C. The time period of the reaction is about 4 hours to about 48hours.

The compound of Formula VIb is reacted with a poly(amino)acidimmunogenic carrier (Z′ of Formula II is KLH, for example) to give acompound of the formula IIb. As shown, R^(1′) is the moiety of compoundof the Formula IIb and R^(1″) and R^(1′″) are each H. However,consistent with the principles described herein, the resulting productmay be a mixture of compounds wherein in one compound of the mixtureR^(1′) is the moiety of the compound of the Formula IIa, and in anothercompound of the mixture R^(1′) and R^(1″) are both the moiety ofcompound of the Formula IIa, and in another compound of the mixture R¹and R^(1′″) are both the moiety of compound of the Formula IIa, and inanother compound of the mixture all three of R^(1′) and R^(1″) andR^(1′″) are the moiety of compound of the Formula IIb. The reaction iscarried out in an organic solvent such as, for example, an alkane (e.g.,hexane or pentane), under conditions for forming an amide. In someexamples the temperature during the reaction is about 15° C. to about25° C. The time period of the reaction is about 4 hours to about 48hours.

In an alternate route, a compound of the Formula IIb may be preparedfrom a compound of the Formula XVIII in a manner similar to thatdescribed above for FIG. 3 for the preparation of the compound of theFormula IIa.

Another example of a method of preparing examples of compounds inaccordance with the principles described herein is described, by way ofillustration and not limitation, with reference to FIG. 7. Otherapproaches may be employed to form the compounds consistent with theprinciples described herein. The preparation of a compound of FormulaXXVII (Y is O in Formula I and w is 0 to 10 or 1 to 10) is set forth inFIG. 7. Referring to FIG. 7, compound of the Formula VII (prepared, forexample, as discussed in FIG. 2) is treated to protect the free hydroxylgroup (R¹⁵ is a protecting group) to form a compound of the FormulaXXIII. The conditions of the reaction are dependent on the nature of theprotecting group, for example.

In one example, compound of the Formula VII is treated withtert-butyldimethylsilyl chloride (by way of example and not limitation)under conditions for forming a silyl ether. The reaction is carried outin a polar organic solvent such as, for example, pyridine,dimethylsulfoxide, an ether (e.g., tetrahydrofuran, ethyl ether, or1,4-dioxane), acetonitrile, dichloromethane, or dimethylformamide (DMF).In some examples the temperature during the reaction is about 15° C. toabout 25° C. The time period of the reaction is about 4 hours to about48 hours.

A compound of the Formula XXIV is formed from the compound of FormulaXXIII by reduction of the ketone group to a hydroxy group, for example.The compound of Formula XXIII may be treated to reduce the hydroxy groupby methods that include, but are not limited to, treatment with a metalhydride such as, for example, LiAlH₄ or NaBH₄, to give the compound ofthe Formula XXIV. The reaction is carried out in an organic solvent suchas, for example, an alkanol (e.g., ethanol). In some examples thetemperature during the reaction is about 0° C. to about 25° C. The timeperiod of the reaction is about 0.5 hours to about 4 hours.

A compound of the Formula XXV is formed from the compound of FormulaXXIV by, for example, reaction with a halo ester (such as, by way ofexample and not limitation) an alpha-haloester in the presence of abase. In this example, the compound of Formula XXV is treated with, forexample, BrCH₂COOR¹⁶ (ethyl alpha-bromoacetate in the example shown, wis 1 and R¹⁶ is ethyl) to give the compound of the Formula XXV. Thereaction is carried out by forming an alkoxide ion from the hydroxygroup of the compound of the Formula XXIV in the presence of a base suchas, for example, KOH, NaOH, Na₂CO₃, or K₂CO₃, and reaction of thealkoxide with the haloester. The solvent for the reaction is an aqueoussolvent, which may contain 1% to 40% of a polar organic solvent such asdescribed above. In some examples the temperature during the reaction isabout 50° C. to about 100° C., or about 50° C. to about 90° C., forexample. The time period of the reaction is about 0.5 hours to about 24hours, or about 1 hour to about 8 hours, for example.

The resulting compound of the Formula XXV is treated to hydrolyze theester group to a carboxylic acid group (de-esterification reaction) togive the compound of the Formula XXVI. Numerous approaches are availablefor de-esterification and include, but are not limited to,saponification or treatment with an aqueous base such as, for example,NaOH or KOH, with heat; acid hydrolysis or treatment with an aqueousacid such as, for example, an aqueous mineral acid (such as, forexample, hydrochloric acid, or sulfuric acid, for example. Theconditions for the reaction are dependent on one or more of the natureof the reagents and the nature of the ester, for example.

A compound of the Formula XXVII is formed by removal of protecting groupR¹⁵ from the compound of Formula XXVI. Various approaches may beemployed for removal of protecting groups and include, but are notlimited to, treatment with dilute mineral acid (such as, for example,hydrochloric acid or sulfuric acid); treatment with an organic acid(such as, for example, acetic acid), in a polar organic solvent (suchas, for example, an alkanol (e.g., methanol or ethanol), an ether (e.g.,ethyl ether, tetrahydrofuran, or 1,4-dioxane), a ketone (e.g., acetone),dimethylsulfoxide, acetonitrile, dichloromethane, dimethylformamide(DMF), or a combination of two or more of the above, which may alsocontain water, under conditions for removing the protecting group toyield the compound of Formula XXVII. The conditions for the reaction aredependent on one or more of the nature of the reagents and the nature ofthe protecting group, for example.

FIG. 8 depicts, by way of illustration and not limitation, an example ofa method of preparing a compound of the Formula VIc and conversion ofthe compound of Formula VIc to a compound of the Formula IIc (where Z′is the enzyme G6PDH, by way of example and not limitation). Referring toFIG. 8, compound of Formula XXVII is treated to activate the carboxylicacid group such as, but not limited to, reaction to form anN-hydroxysuccinimide (NHS) ester (compound of the Formula XXVIII). Thereaction is carried out in a polar organic solvent such as, for example,dimethylsulfoxide, an ether (e.g., tetrahydrofuran, ethyl ether, or1,4-dioxane), acetonitrile, dichloromethane, or dimethylformamide (DMF),for example, under conditions for forming an NHS ester. In some examplesthe temperature during the reaction is about 15° C. to about 25° C. Thetime period of the reaction is about 1 hour to about 24 hours.

NHS ester of the Formula XXVIII is combined with a compound of theFormula XXI to form compound of the formula VIc. The reaction is carriedout in an organic solvent such as, for example, an alkane (e.g., hexaneor pentane), for example, under conditions for forming an amide. In someexamples the temperature during the reaction is about 15° C. to about25° C. The time period of the reaction is about 1 hour to about 24hours.

The compound of Formula VIc is reacted with the enzyme, G6PDH (Z′ ofFormula II is G6PDH, for example), to give a compound of the FormulaIIc. As shown, R^(1′) is the moiety of compound of the Formula IIc andR^(1″) and R^(1′″) are each H. However, consistent with the principlesdescribed herein, the resulting product may be a mixture of compoundswherein in one compound of the mixture R^(1′) is the moiety of thecompound of the Formula IIa, and in another compound of the mixtureR^(1′) and R^(1″) are both the moiety of compound of the Formula IIa,and in another compound of the mixture R¹ and R^(1′″) are both themoiety of compound of the Formula IIa, and in another compound of themixture all three of R¹ and R^(1″) and R^(1′″) are the moiety ofcompound of the Formula IIc. The reaction is carried out in an organicsolvent such as, for example, an alkane (e.g., hexane or pentane), underconditions for forming an amide. In some examples the temperature duringthe reaction is about 15° C. to about 25° C. The time period of thereaction is about 1 hour to about 24 hours.

In an alternate route, a compound of the Formula IIc may be preparedfrom a compound of the Formula XXVII in a manner similar to thatdescribed above for FIG. 3 for the preparation of the compound of theFormula IIa.

Other compounds in accordance with the principles described herein maybe prepared in a manner similar to that described above.

Preparation of Binding Partners

Examples of compounds in accordance with the principles described hereinwhere Z is a poly(amino acid) immunogenic carrier or a non-poly(aminoacid) immunogenic carrier may be employed to prepare binding partnersfor vitamin D such as, for example, aptamers for vitamin D or antibodiesfor vitamin D, which include, but are not limited to, antibodiesspecific for vitamin D₃, antibodies specific for vitamin D₂, antibodiesspecific for 25-hydroxyvitamin D₃, antibodies specific for25-hydroxyvitamin D₂, antibodies specific for 3-epi-25-hydroxyvitaminD₃, and antibodies specific for 3-epi-25-hydroxyvitamin D₂, for example.Of particular interest are antibodies specific for3-epi-25-hydroxyvitamin D₃, antibodies specific for3-epi-25-hydroxyvitamin D₂, and antibodies specific for epimers of othervitamin D compounds (“antibodies for epimeric vitamin D”), which can beemployed in assays for 3-epi-25-hydroxyvitamin D₃ and for3-epi-25-hydroxyvitamin D₂, or which can be employed in assays fornon-epimeric vitamin D to reduce or eliminate interference from epimericforms of vitamin D such as, for example, 3-epi-25-hydroxyvitamin D₃ andfrom 3-epi-25-hydroxyvitamin D₂, which may be present in a sample to betested for the presence of vitamin D.

Antibodies may be a monoclonal antibodies or a polyclonal antibodies andmay include a complete immunoglobulin or fragment thereof, whichimmunoglobulins include, but are not limited to, various classes andisotypes, such as IgA, IgD, IgE, IgG and IgM, for example. Fragmentsthereof may include Fab, Fv and F(ab′)₂, Fab′, and the like. Inaddition, aggregates, polymers, and conjugates of immunoglobulins ortheir fragments can be used where appropriate so long as bindingaffinity for a particular molecule is maintained.

Antibodies in accordance with the principles described herein may beprepared by techniques including, but not limited to, immunization of ahost and collection of sera (polyclonal), preparing continuous hybridcell lines and collecting the secreted protein (monoclonal) or cloningand expressing nucleotide sequences or mutagenized versions thereofcoding at least for the amino acid sequences required for specificbinding of natural antibodies, for example.

Monoclonal antibodies can be prepared by techniques such as preparingcontinuous hybrid cell lines and collecting the secreted protein(somatic cell hybridization techniques). Monoclonal antibodies may beproduced according to the standard techniques of Köhler and Milstein,Nature 265:495-497, 1975. Reviews of monoclonal antibody techniques arefound in Lymphocyte Hybridomas, ed. Melchers, et al. Springer-Verlag(New York 1978), Nature 266: 495 (1977), Science 208: 692 (1980), andMethods of Enzymology 73 (Part B): 3-46 (1981).

In another approach for the preparation of antibodies, the sequencecoding for antibody binding sites can be excised from the chromosome DNAand inserted into a cloning vector, which can be expressed in bacteriato produce recombinant proteins having the corresponding antibodybinding sites. This approach involves cloning and expressing nucleotidesequences or mutagenized versions thereof coding at least for the aminoacid sequences required for specific binding of natural antibodies.

In one approach for the production of monoclonal antibodies, a firststep includes immunization of an antibody-producing animal such as amouse, a rat, a goat, a sheep, or a cow with an immunogen that comprisesa compound of Formula I wherein Z is an immunogenic carrier, forexample. Immunization can be performed with or without an adjuvant suchas complete Freund's adjuvant or other adjuvants such as monophosphoryllipid A and synthetic trehalose dicorynomycolate adjuvant. A next stepincludes isolating spleen cells from the antibody-producing animal andfusing the antibody-producing spleen cells with an appropriate fusionpartner, typically a myeloma cell, such as by the use of polyethyleneglycol or other techniques. Typically, the myeloma cells used are thosethat grow normally in hypoxanthine-thymidine (HT) medium but cannot growin hypoxanthine-aminopterin-thymidine (HAT) medium, used for selectionof the fused cells. A next step includes selection of the fused cells,typically by selection in HAT medium. A next step includes screening thecloned hybrids for appropriate antibody production using immunoassayssuch as, for example, an enzyme-linked immunosorbent assay (ELISA) orother immunoassays appropriate for screening.

An antibody (prepared from an immunogen in accordance with theprinciples described herein) with the requisite specificity may beselected by screening methodologies, which include, by way ofillustration and not limitation, ELISA, dot blots, Western analysis, andSurface Plasmon Resonance, for example. In this manner an antibody isobtained that binds to a vitamin D analyte of interest and does not bindto any detectable degree to a vitamin D molecule that is not of interestin a particular assay. In some examples in accordance with theprinciples described herein, an antibody that binds to a vitamin Danalyte of interest has a binding affinity for the vitamin D analyte ofinterest of about 10⁷ to about 10¹⁴ liters/mole, or about 10⁷ to about10¹¹ liters/mole, or about 10⁷ to about 10¹² liters/mole, or about 10⁸to about 10¹⁴ liters/mole, or about 10⁸ to about 10¹¹ liters/mole, orabout 10⁸ to about 10¹² liters/mole, for example. The phrase “anydetectable degree” means that the antibody that specifically binds to avitamin D analyte of interest (e.g., 3-3pi-25-hydroxyvitamin D) has abinding affinity for a vitamin D molecule that is not of interest (e.g.,a non-epi-vitamin D compound) of less than about 10⁴ liters/mole, orless than about 10³ liters/mole, or less than about 10² liters/mole, orless than about 10 liters/mole, for example.

In one example in accordance with the principles described herein, animmunogen, prepared from a compound of Formula I above wherein Z is animmunogenic carrier, is employed to prepare antibodies that are specificfor 3-epi-25-hydroxyvitamin D₃ where the antibodies do not bind to anydetectable degree with 25-hydroxyvitamin D₃ or to other variants ofvitamin D including non-epi-vitamin D compounds such as, for example,25-hydroxyvitamin D; calcidiol; 1,25-dihydroxyvitamin D₃; 1,25-dihydroxyvitamin D₄; 1,25-dihydroxy vitamin D₅; and 1,25-dihydroxy vitamin D₆,for example.

In another example in accordance with the principles described herein,an immunogen, prepared from a compound of Formula I above wherein Z isan immunogenic carrier, is employed to prepare antibodies that arespecific for 3-epi-25-hydroxyvitamin D₂ where the antibodies do not bindto any detectable degree with 25-hydroxyvitamin D₂ or to other variantsof vitamin D including non-epi-vitamin D compounds such as, for example,25-hydroxyvitamin D; calcidiol; 1,25-dihydroxyvitamin D₃; 1,25-dihydroxyvitamin D₄; 1,25-dihydroxy vitamin D₅; and 1,25-dihydroxy vitamin D₆,for example.

In one example, by way of illustration and not limitation, an immunogenis employed wherein Z in the compound of Formula I is BSA. Thisimmunogen is used to immunize mice (e.g., BALB/c mice, Swiss Webstermice or an AJ strain of mice) intraperitoneally. Serum samples fromthese mice are tested for anti-3-epi-25-hydroxyvitamin D₃ antibodiesusing a conjugate of 3-epi-25-hydroxyvitamin D₃ and ovalbumin (ovalbuminconjugate). A microtiter plate ELISA is employed and the antibodies areexamined for binding to the ovalbumin conjugate and subsequently to3-epi-25-hydroxyvitamin D₃. Mice with highest titers are boosted threedays prior to fusion. On the day of fusion, spleen cells are harvestedfrom these mice and are fused with myeloma cell line P3X63Ag8.653 usingPEG assisted fusion protocols. After about ten days, hybridomassupernatants are screened for anti-3-epi-25-hydroxyvitamin D₃ antibodiesusing a plate ELISA. Positive clones are further expanded, sub-clonedand supernatants are purified using a protein A sepharose column.Purified antibody samples are tested using ELISA for binding to theovalbumin conjugate and to free 3-epi-25-hydroxyvitamin D₃.

Particular examples, by way of illustration and not limitation, ofantibodies prepared as described above that are specific for3-epi-25-hydroxyvitamin D₃ or specific for 3-epi-25-hydroxyvitamin D₂,include antibody 4G8 and antibody 8F10, for example.

In some examples, binding partners such as, for example, antibodies inaccordance with the principles described herein may be employed topurify vitamin D compounds. For example, antibodies such as thosedescribed above may be bound to a support and the support employed topurify vitamin D compounds. In one example, antibodies for3-epi-25-hydroxyvitamin D₃ may be bound to an affinity purificationchromatography substrate such as, for example, a column, and vitamin Dpreparations may be contacted with the chromatography substrate whereantibody on the substrate binds 3-epi-25-hydroxyvitamin D₃ from thevitamin D preparation while other vitamin D compounds are eluted fromthe substrate.

General Description of Assays for Vitamin D

Some examples in accordance with the principles described herein aredirected to methods of determining one or both of the presence and theamount of a vitamin D analyte in a sample suspected of containing thevitamin D analyte and may be referred to herein as “assays for vitaminD.”

The phrase “vitamin D” refers to one or more of 25-hydroxyvitamin D;calcidiol; 1,25-dihydroxy vitamin D₂; 1,25-dihydroxyvitamin D₃;1,25-dihydroxy vitamin D₄; 1,25-dihydroxy vitamin D₅; and 1,25-dihydroxyvitamin D₆; including epimeric forms and metabolites of all of theabove. Thus, vitamin D analyte includes vitamin D and epimers of vitaminD as defined above. Some examples in accordance with the principlesdescribed herein are directed to methods of determining one or both ofthe presence and the amount of epimeric forms of vitamin D in a samplesuspected of containing epimeric forms of vitamin D (such as, forexample, 3-epi-25-hydroxyvitamin D₃ or 3-epi-25-hydroxyvitamin D₂) andmay be referred to herein as “assays for epimers of vitamin D.”

In an example, by way of illustration and not limitation, of method fordetermining a vitamin D analyte, a combination is provided thatcomprises the sample, a binding partner such as, for example, anantibody for an epimer of vitamin D produced in accordance with theprinciples described herein, a binding partner such as, for example, anantibody for the vitamin D analyte and a conjugate comprising a vitaminD analog and a member of a signal producing system.

As mentioned above, the sample and reagents are provided “in combinationin the medium.” While the order of addition to the medium may be variedto form the combination, there will be certain preferences for someembodiments of the assay formats described herein. In one example, byway of illustration and not limitation, the order of addition is to addall the materials simultaneously and determine the effect that the assaymedium has on the signal as in a homogeneous assay. In another example,by way of illustration and not limitation, each of the reagents, orgroups of reagents, can be combined sequentially. In some embodiments,an incubation step may be involved subsequent to each addition asdiscussed above. In heterogeneous assays, separation and washing stepsmay also be employed after one or more incubation steps.

The phrase “vitamin D analog” refers to a compound that competes with avitamin D analyte for a receptor such as an antibody for vitamin D or anantibody for a epimer of vitamin D. The vitamin D analog may be amodified vitamin D where the modification provides means to join vitaminD to another molecule such as, but not limited to, a support, a label, asmall molecule, or a binding partner for a small molecule, for example.The vitamin D analog may be linked to another molecule directly orindirectly by means of a linking group. The vitamin D analog may be, forexample, a molecule structurally related to vitamin D or vitamin Dconjugated to another molecule through a linking group. In some examplesin accordance with the principles described herein, a vitamin D analogmay be a compound of the Formula I wherein Z is poly(amino acid) labelmoiety or a non-poly(amino acid) label moiety or wherein Z is animmunogenic carrier where such compound of Formula I competes with avitamin D analyte for binding to a vitamin D antibody.

The sample to be analyzed is one that is suspected of containing avitamin D analyte. The samples may be biological samples ornon-biological samples. Biological samples may be from a mammaliansubject or a non-mammalian subject. Mammalian subjects may be, e.g.,humans or other animal species. Biological samples include biologicalfluids such as whole blood, serum, plasma, sputum, lymphatic fluid,semen, vaginal mucus, feces, urine, spinal fluid, saliva, stool,cerebral spinal fluid, tears, mucus, and the like; biological tissuesuch as hair, skin, sections or excised tissues from organs or otherbody parts; and so forth. In many instances, the sample is whole blood,plasma or serum. Non-biological samples including, but not limited to,waste streams, for example, may also be analyzed using compounds inaccordance with the principles described herein.

The sample can be prepared in any convenient medium, which may be, forexample, an assay medium, which is discussed more fully hereinbelow. Insome instances a pretreatment may be applied to the sample such as, forexample, to lyse blood cells. In some examples, such pretreatment isperformed in a medium that does not interfere subsequently with anassay.

The combination in the medium is subjected to conditions for binding ofthe vitamin D analyte and the vitamin D analog to the antibody forvitamin D analyte to form a complex. The amount of the complex ismeasured where the amount of the complex is related to one or both ofthe presence and amount of the vitamin D analyte in the sample.

An assay for a vitamin D analyte can be performed either withoutseparation (homogeneous) or with separation (heterogeneous) of any ofthe assay components or products. Heterogeneous assays usually involveone or more separation steps and can be competitive or non-competitive.Immunoassays may involve labeled or non-labeled reagents. Immunoassaysinvolving non-labeled reagents usually comprise the formation ofrelatively large complexes involving one or more antibodies preparedfrom immunogenic conjugates in accordance with the principles describedherein. Such assays include, for example, immunoprecipitin andagglutination methods and corresponding light scattering techniques suchas, e.g., nephelometry and turbidimetry, for the detection of antibodycomplexes. Labeled immunoassays include, but are not limited to,chemiluminescence immunoassays, enzyme immunoassays, fluorescencepolarization immunoassays, radioimmunoassays, inhibition assays, inducedluminescence assays, and fluorescent oxygen channeling assays, forexample.

One general group of immunoassays includes immunoassays using a limitedconcentration of a compound in accordance with the principles describedherein. Another group of immunoassays involves the use of an excess ofone or more of the principal reagents such as, for example, an excess ofa compound in accordance with the principles described herein. Anothergroup of immunoassays includes separation-free homogeneous assays inwhich signal from a labeled vitamin D analog is modulated upon bindingof the labeled vitamin D analog to an antibody produced in accordancewith the principles described herein, thus competing with a vitamin Danalyte that may be present in the sample.

As mentioned above, the assays can be performed either withoutseparation (homogeneous) or with separation (heterogeneous) of any ofthe assay components or products. Homogeneous immunoassays areexemplified by the EMIT® assay (Siemens Healthcare Diagnostics Inc.,Deerfield, Ill.) disclosed in Rubenstein, et al., U.S. Pat. No.3,817,837, column 3, line 6 to column 6, line 64; immunofluorescencemethods such as those disclosed in Ullman, et al., U.S. Pat. No.3,996,345, column 17, line 59, to column 23, line 25; enzyme channelingimmunoassays (“ECIA”) such as those disclosed in Maggio, et al., U.S.Pat. No. 4,233,402, column 6, line 25 to column 9, line 63; thefluorescence polarization immunoassay (“FPIA”) as disclosed, forexample, in, among others, U.S. Pat. No. 5,354,693; and enzymeimmunoassays such as the enzyme linked immunosorbant assay (“ELISA”).Exemplary of heterogeneous assays are the radioimmunoassay, disclosed inYalow, et al., J. Clin. Invest. 39:1157 (1960). The relevant portions ofthe above disclosures are all incorporated herein by reference.

Other enzyme immunoassays are the enzyme modulate mediated immunoassay(“EMMIA”) discussed by Ngo and Lenhoff, FEBS Lett. (1980) 116:285-288;the substrate labeled fluorescence immunoassay (“SLFIA”) disclosed byOellerich, J. Clin. Chem. Clin. Biochem. (1984) 22:895-904; the combinedenzyme donor immunoassays (“CEDIA”) disclosed by Khanna, et al., Clin.Chem. Acta (1989) 185:231-240; homogeneous particle labeled immunoassayssuch as particle enhanced turbidimetric inhibition immunoassays(“PETINIA”), particle enhanced turbidimetric immunoassay (“PETIA”); theAffinity Chromium dioxide Mediated Immuno Assay (“ACMIA”) assay format,which is described in U.S. Pat. Nos. 7,186,518, 5,147,529, 5,128,103,5,158,871, 4,661,408, 5,151,348, 5,302,532, 5,422,284, 5,447,870, and5,434,051, the disclosures of which are incorporated herein in theirentirety; for example.

Other assays include acridinium ester label assays such as thosediscussed in U.S. Pat. Nos. 6,355,803; 6,673,560; 7,097,995 and7,319,041, the relevant disclosures of which are incorporated herein byreference. A particular example of an acridinium ester label assay is anacridinium ester label immunoassay using paramagnetic particles as asolid phase (“ADVIA” immunoassay). Other assays include the sol particleimmunoassay (“SPIA”), the disperse dye immunoassay (“DIA”); themetalloimmunoassay (“MIA”); the enzyme membrane immunoassays (“EMIA”);and luminoimmunoassays (“LIA”). Other types of assays includeimmunosensor assays involving the monitoring of the changes in theoptical, acoustic and electrical properties of the present conjugateupon the binding of vitamin D analyte. Such assays include, for example,optical immunosensor assays, acoustic immunosensor assays, semiconductorimmunosensor assays, electrochemical transducer immunosensor assays,potentiometric immunosensor assays, amperometric electrode assays.

Heterogeneous assays usually involve one or more separation steps andcan be competitive or non-competitive. A variety of competitive andnon-competitive heterogeneous assay formats are disclosed in Davalian,et al., U.S. Pat. No. 5,089,390, column 14, line 25 to column 15, line9, incorporated herein by reference. In an example of a competitiveheterogeneous assay, a support having an antibody for a vitamin Danalyte bound thereto is contacted with a medium containing the samplesuspected of containing the vitamin D analyte and a labeled compound inaccordance with the principles described herein. Vitamin D analyte inthe sample competes, for binding to the antibody for the vitamin Danalyte, with a vitamin D analog bearing a detectable label. Afterseparating the support and the medium, the label activity of the supportor the medium is determined by conventional techniques and is related tothe amount of vitamin D analyte in the sample. In a variation of theabove competitive heterogeneous assay, the support comprises a vitamin Danalog as the labeled reagent and vitamin D antibody comprises a label.

In some examples, a sample to be analyzed is combined in an assay mediumwith an antibody for the vitamin D analyte and labeled vitamin D analog.The medium is examined for one or both of the presence and amount of acomplex comprising the labeled vitamin D analog and the antibody for thevitamin D analyte where the presence and/or the amount of such complexindicates the presence and/or amount of the vitamin D analyte in thesample.

In some examples in accordance with the principles described herein, thesample to be analyzed is subjected to a pretreatment to release thevitamin D analyte from endogenous binding substances such as, forexample, plasma or serum proteins that bind vitamin D. The release ofthe vitamin D analyte from endogenous binding substances may be carriedout, for example, by addition of a digestion agent or a releasing agentor a combination of a digestion agent and a releasing agent usedsequentially. The digestion agent is one that breaks down the endogenousbinding substances so that they can no longer bind vitamin D. Suchagents include, but are not limited to, proteinase K and proteinase Kand protein denaturing agents such as, e.g., detergents (sodium dodecylsulfate, for example). Releasing agents for releasing vitamin D fromendogenous binding substances include, by way of illustration and notlimitation, acidic denaturing agents such as, for example, salicylicacid, warfarin, sulfonic acids, toluene sulfonic acids, naphthalenesulfonic acid, anilinonaphthalene sulfonic acids (ANS) (including, e.g.,1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) and8-anilinonaphthalene-1-sulfonic acid (8-ANS)), salicylic acids andderivatives and salts of the above.

The conditions such as, for example, duration, temperature, pH andconcentration of the releasing agent in the medium for carrying out thedigestion or releasing actions are dependent on the nature of theendogenous binding substances, the nature of the sample, and the natureof the releasing agent, for example. In general, the conditions aresufficient to achieve the desired effect or function. In some examplesin accordance with the principles described herein, an effectiveconcentration of releasing agent is about 0.01 to about 20 mg/mL, orabout 0.01 to about 10 mg/mL, or about 0.01 to about 5 mg/mL, or about0.1 to about 20 mg/mL, or about 0.1 to about 10 mg/mL, or about 0.1 toabout 5 mg/mL, or about 0.1 to about 1 mg/mL. The pretreatment of thesample to release the vitamin D analyte from endogenous bindingsubstances may be carried out as a separate step prior to conducting anassay or as a first step in an assay. In either case, one or morereagents may be required to stop the action of the digestion agentand/or the releasing agent.

The conditions for conducting the assays include carrying out the assayin an aqueous buffered medium at a moderate pH, generally that whichprovides optimum assay sensitivity. The aqueous medium may be solelywater or may include from 0.1 to about 40 volume percent of a cosolvent.The pH for the medium will be in the range of about 4 to about 11, or inthe range of about 5 to about 10, or in the range of about 6.5 to about9.5, for example. The pH will usually be a compromise between optimumbinding of the binding members of any specific binding pairs, the pHoptimum for other reagents of the assay such as members of the signalproducing system, and so forth. Various buffers may be used to achievethe desired pH and maintain the pH during the assay. Illustrativebuffers include, by way of illustration and not limitation, borate,phosphate, carbonate, TRIS, barbital, PIPES, HEPES, MES, ACES, MOPS, andBICINE, for example. The particular buffer employed is not critical, butin an individual assay one or another buffer may be preferred.

Various ancillary materials may be employed in the assay methods. Forexample, in addition to buffers the medium may comprise stabilizers forthe medium and for the reagents employed. In some embodiments, inaddition to these additives, proteins may be included, such as, forexample, albumins; organic solvents such as, for example, formamide;quaternary ammonium salts; polyanions such as, for example, dextransulfate; binding enhancers, for example, polyalkylene glycols;polysaccharides such as, for example, dextran or trehalose. The mediummay also comprise agents for preventing the formation of blood clots.Such agents are well known in the art and include, but are not limitedto, EDTA, EGTA, citrate, heparin, for example. The medium may alsocomprise one or more preservatives such as, but not limited to, sodiumazide, neomycin sulfate, PROCLIN® 300, Streptomycin, for example. Themedium may additionally comprise one or more surfactants. Any of theabove materials, if employed, is present in a concentration or amountsufficient to achieve the desired effect or function.

One or more incubation periods may be applied to the medium at one ormore intervals including any intervals between additions of variousreagents employed in an assay including those mentioned above. Themedium is usually incubated at a temperature and for a time sufficientfor binding of various components of the reagents and binding of vitaminD in the sample to occur. Moderate temperatures are normally employedfor carrying out the method and usually constant temperature,preferably, room temperature, during the period of the measurement. Insome examples, incubation temperatures range from about 5° to about 99°C., or from about 15° C. to about 70° C., or from about 20° C. to about45° C., for example. The time period for the incubation, in someexamples, is about 0.2 seconds to about 24 hours, or about 1 second toabout 6 hours, or about 2 seconds to about 1 hour, or about 1 minute toabout 15 minutes, for example. The time period depends on thetemperature of the medium and the rate of binding of the variousreagents, which is determined by the association rate constant, theconcentration, the binding constant and dissociation rate constant.

In an example of a method for determining a vitamin D analyte in asample suspected of containing the vitamin D analyte, a combination isprovided in a medium where the combination includes the sample, areleasing agent (if the sample has not been pretreated to release thevitamin D analyte from endogenous binding substances), an antibody forvitamin D, and a labeled vitamin D analog where the label is apoly(amino acid) label or a non-poly(amino acid) label. The medium isexamined for one or both of the presence and amount of one or both of acomplex comprising vitamin D and the antibody for vitamin D or a complexcomprising the labeled compound and antibody for vitamin D. The presenceand/or the amount of one or both of the complexes indicates the presenceand/or amount of the vitamin D analyte in the sample.

Some known assays utilize a signal producing system (sps) that employsfirst and second sps members. The designation “first” and “second” iscompletely arbitrary and is not meant to suggest any order or rankingamong the sps members or any order of addition of the sps members in thepresent methods. The sps members may be related in that activation ofone member of the sps produces a product such as, e.g., light or anactivated product, which results in activation of another member of thesps.

In some embodiments of assays, the sps members comprise a sensitizersuch as, for example, a photosensitizer, and a chemiluminescentcomposition where activation of the sensitizer results in a product thatactivates the chemiluminescent composition. The second sps memberusually generates a detectable signal that relates to the amount ofbound and/or unbound sps member, i.e., the amount of sps member bound ornot bound to the vitamin D analyte being detected or to a compound inaccordance with the principles described herein. In some examples inaccordance with the principles described herein, one of either thesensitizer reagent or the chemiluminescent reagent comprises the presentcompound reagent.

In a particular example, an induced luminescence immunoassay may beemployed. The induced luminescence immunoassay is referred to in U.S.Pat. No. 5,340,716 (Ullman), which disclosure is incorporated herein byreference. In one approach, the assay uses a particle having associatedtherewith a photosensitizer where a vitamin D analog is bound to theparticle (particle-compound reagent). The chemiluminescent reagentcomprises an antibody for the vitamin D analyte. The vitamin D analytecompetes with the particle-compound reagent for binding to the antibodyfor vitamin D. If the vitamin D analyte is present, the fewer is thenumber of molecules of particle-compound reagent that come into closeproximity with the chemiluminescent reagent. Therefore, there will be adecrease in the assay signal. The photosensitizer generates singletoxygen and activates the chemiluminescent reagent when the two labelsare in close proximity. The activated chemiluminescent reagentsubsequently produces light. The amount of light produced is related tothe amount of the complex formed, which in turn is related to the amountof vitamin D analyte present in the sample.

In another particular example of an induced luminescence immunoassay,the assay uses a particle having associated therewith a chemiluminescentcompound where a vitamin D analog is bound to the particle(particle-compound reagent). The photosensitizer reagent comprises anantibody for vitamin D. The vitamin D analyte competes with theparticle-compound reagent for binding to the antibody for vitamin D. Ifthe vitamin D analyte is present, the fewer is the number of moleculesof particle-compound reagent that come into close proximity with thephotosensitizer reagent. Therefore, there will be a decrease in theassay signal. The photosensitizer generates singlet oxygen and activatesthe chemiluminescent compound of the particle-compound reagent when thetwo labels are in close proximity. The activated chemiluminescentcompound subsequently produces light. The amount of light produced isrelated to the amount of the complex formed, which in turn is related tothe amount of vitamin D analyte present in the sample.

In another particular example of an induced luminescence assay, aphotosensitizer particle is employed that is conjugated to a bindingpartner for a small molecule such as, for example, avidin orstreptavidin (which are binding partners for biotin). A vitamin D analogthat comprises biotin (compound-biotin reagent) is also employed. Achemiluminescent reagent that comprises an antibody for the vitamin Danalyte is employed as part of the detection system. The reaction mediumis incubated to allow the avidin or streptavidin of the photosensitizerparticles to bind to the compound-biotin reagent by virtue of thebinding between avidin and biotin and to also allow the antibody for thevitamin D analyte that is part of the chemiluminescent reagent to bindto the vitamin D analyte or to vitamin D analog that is now attached tothe photosensitizer particles. Then, the medium is irradiated with lightto excite the photosensitizer, which is capable in its excited state ofactivating oxygen to a singlet state. Because less of thechemiluminescent reagent is now in close proximity to thephotosensitizer because of the presence of the vitamin D analyte, thereis less activation of the chemiluminescent reagent by the singlet oxygenand less luminescence. The medium is then examined for the presenceand/or the amount of luminescence or light emitted, the presence thereofbeing related to the presence and/or amount of the vitamin D analytewhere a decrease in signal is observed in the presence of the vitamin Danalyte.

In another particular example of an induced luminescence assay, aphotosensitizer particle is employed that is conjugated to a bindingpartner for a small molecule such as, for example, avidin orstreptavidin (which are binding partners for biotin). A conjugatereagent comprises an antibody for the vitamin D analyte conjugated tobiotin. A vitamin D analog is employed where the compound is attached toa chemiluminescent particle (chemiluminescent-compound reagent) is alsoemployed. The reaction medium is incubated to allow the avidin orstreptavidin of the photosensitizer particles to bind to theantibody-biotin reagent by virtue of the binding between avidin andbiotin and to also allow antibody for the vitamin D analyte to bind tothe vitamin D analyte if present in the sample and to the vitamin Danalog that is part of the chemiluminescent-compound reagent. Then, themedium is irradiated with light to excite the photosensitizer, which iscapable in its excited state of activating oxygen to a singlet state.Because less of the chemiluminescent-compound reagent is now in closeproximity to the photosensitizer because of the presence of the vitaminD analyte, there is less activation of the chemiluminescent reagent bythe singlet oxygen and less luminescence. The medium is then examinedfor the presence and/or the amount of luminescence or light emitted, thepresence thereof being related to the presence and/or amount of thevitamin D analyte where a decrease in signal is observed in the presenceof the vitamin D analyte.

Another example, by way of illustration and not limitation, of an assayformat for detection of a vitamin D analyte is the ACMIA assay format.For the ACMIA assay format, chrome particles, which are coated with avitamin D analog (chrome particle reagent), are employed as a firstcomponent. A second component is an antibody for the vitamin D analyte.This antibody, crosslinked to a reporter enzyme (for example,β-galactosidase) to form an antibody-enzyme conjugate, is added to areaction vessel in an excess amount, i.e., an amount greater than thatrequired to bind all of the vitamin D analyte that might be present in asample. A sample, which is previously subjected to treatment with areleasing agent, is treated with an antibody for the vitamin D analyte,which binds to the vitamin D analyte in the sample. The antibody-enzymeconjugate is mixed with sample in the medium to allow the vitamin Danalyte to bind to the antibody. Next, the chrome particle reagent isadded to bind up any excess antibody-enzyme conjugate. Then, a magnet isapplied, which pulls all of the chrome particles and excessantibody-enzyme out of the suspension, and the supernatant istransferred to a final reaction container. The substrate of the reporterenzyme is added to the final reaction container, and the enzyme activityis measured spectrophotometrically as a change in absorbance over time.The amount of this signal is related to the amount of the vitamin Danalyte in the sample.

Another example of an assay for vitamin D in accordance with theprinciples described herein is an acridinium ester label immunoassayusing paramagnetic particles as a solid phase (ADVIA immunoassay). Thedetection system employed for this example of a vitamin D assay includesa small molecule-labeled vitamin D analog (capture moiety) as the smallmolecule conjugate or capture conjugate, binding partner for the smallmolecule-coated paramagnetic latex particles as a solid phase (SP), andan acridinium ester labeled antibody for the vitamin D analyte(detection antibody). The small molecule may be, for example, biotin orfluorescein and the respective binding partner may be streptavidin orantibody for fluorescein. The vitamin D analog may be linked to thesmall molecule directly or through a linking group such as, for example,a protein, e.g., bovine serum albumin (BSA). Vitamin D analyte in apatient sample competes with vitamin D analog of the capture moiety forbinding to the acridinium ester labeled detection anti-vitamin Dantibody. The sample suspected of containing vitamin D is subjected to apretreatment with 1,8-ANS. The assay may be carried out on a Centaur®,Centaur® XP or Centaur® CP apparatus (Siemens Healthcare DiagnosticsInc., Newark Del.) in accordance with the manufacturer's directionssupplied therewith.

Another example of an assay for a vitamin D analyte in accordance withthe principles described herein is an acridinium ester label immunoassayusing paramagnetic particles as a solid phase (ADVIA immunoassay). Thedetection system employed for this example of a vitamin D assay includesa small molecule-labeled antibody for the vitamin D analyte (captureantibody) as the biotin conjugate or capture conjugate,streptavidin-coated paramagnetic latex particles as a solid phase (SP),and an acridinium ester labeled vitamin D analog (detection hapten). Theacridinium ester label may be directly bound to the vitamin D analog toform the detection hapten or a linking group may be employed including,for example, a protein such as, e.g., BSA. Vitamin D analyte in apatient sample competes with the acridinium ester labeled detectionhapten for binding with anti-vitamin D antibody. The sample suspected ofcontaining vitamin D is subjected to a pretreatment with 1,8-ANS. Theassay may be carried out on a Centaur®, Centaur® XP or Centaur® CPapparatus (Siemens Healthcare Diagnostics Inc., Newark Del.) inaccordance with the manufacturer's directions supplied therewith. Invariations of the above acridinium ester assays, the small molecule maybe, for example, biotin or fluorescein.

The concentration of the vitamin D analyte in a sample that may beassayed generally varies from about 10⁻⁵ to about 10⁻¹⁷ M, or from about10⁻⁶ to about 10⁻¹⁴ M, for example. Considerations such as whether theassay is qualitative, semi-quantitative or quantitative (relative to theamount of the vitamin D analyte present in the sample), the particulardetection technique and the expected concentration of the vitamin Danalyte normally determine the concentrations of the various reagents.

The concentrations of the various reagents in the assay medium willgenerally be determined by the concentration range of interest of thevitamin D analyte, the nature of the assay, and the like. However, thefinal concentration of each of the reagents is normally determinedempirically to optimize the sensitivity of the assay over the range ofinterest. That is, a variation in concentration of vitamin D analytethat is of significance should provide an accurately measurable signaldifference. Considerations such as the nature of the signal producingsystem and the nature of the analytes normally determine theconcentrations of the various reagents.

Examples of Assays for Epimers of Vitamin D Employing Reagents inAccordance with the Principles Described Herein

As mentioned above, one particular example in accordance with theprinciples described herein is directed to methods of determining one orboth of the presence and the amount of epimers of vitamin D in a samplesuspected of containing epimers of vitamin D. Any of the above assayformats may be employed using an antibody raised against an immunogenthat is a compound of the Formula I wherein Z is an immunogenic carrier.In some examples the immunogen is a compound of the Formula IIa (seeFIG. 3). In some examples the immunogen is a compound of the Formula IIb(see FIG. 5). In some examples, the antibody employed is antibody 4G8 orantibody 8F10. Antibodies produced from other immunogens consistent withthe principles described herein may be employed in any of the aboveassay formats.

One example of an assay for an epimer of vitamin D is an inducedluminescence assay for the detection of 3-epi-25-hydroxyvitamin D₃ in asample suspected of containing 3-epi-25-hydroxyvitamin D₃. The sample istreated with a releasing agent to release 3-epi-25-hydroxyvitamin D₃from endogenous binding moieties. Then, the sample is combined with areagent that comprises antibody 4G8.2, which is an antibody for3-epi-25-hydroxyvitamin D₃, conjugated to biotin (antibody-biotinreagent) in a reaction medium, which is incubated to allow the bindingbetween the antibody-biotin reagent and the 3-epi-25-hydroxyvitamin D₃analyte. Then, a chemiluminescent reagent is added where the reagentcomprises a compound of the Formula IIa as a vitamin D analog attachedto a chemiluminescent particle. The chemiluminescent reagent binds toexcess antibody-biotin reagent, that is, antibody-biotin reagent thatdoes not bind to 3-epi-25-hydroxyvitamin D₃ analyte in the sample. Then,a photosensitizer particle reagent is added that is conjugated to astreptavidin. The reaction medium is incubated to allow the streptavidinof the photosensitizer particles to bind to the antibody-biotin reagentby virtue of the binding between streptavidin and biotin. Then, themedium is irradiated with light to excite the photosensitizer, which iscapable in its excited state of activating oxygen to a singlet state.Because less of the chemiluminescent reagent is now in close proximityto the photosensitizer because of the presence of the3-epi-25-hydroxyvitamin D₃ analyte, there is less activation of thechemiluminescent reagent by the singlet oxygen and less luminescence.The medium is then examined for the presence and/or the amount ofluminescence or light emitted, the presence thereof being related to thepresence and/or amount of the 3-epi-25-hydroxyvitamin D₃ analyte where adecrease in signal is observed in the presence of the3-epi-25-hydroxyvitamin D₃ analyte.

Examples of Assays for Vitamin D Analytes Employing Antibodies inAccordance with the Principles Described Herein as Blocking Antibodies

Antibodies in accordance with the principles described herein may beemployed to minimize or eliminate 3-epimer cross-reactivity in assaysfor non-epimeric forms of vitamin D analytes. Over-estimation of totalnon-epimeric vitamin D analyte caused by the cross-reactivity of3-epimer vitamin D with an antibody for vitamin D analyte can besubstantially avoided employing, as blocking agents, antibodies preparedagainst immunogens that are a compound of the Formula I wherein Z is animmunogenic carrier.

In one example of an assay for non-epimeric vitamin D analyte, the assayuses, as a chemiluminescent reagent, a chemibead reagent that comprisesan olefinic dye and 25-hydroxyvitamin D₃ as a vitamin D analog. A samplesuspected of containing non-epimeric vitamin D analyte is combined in anassay medium with a biotinylated antibody for the non-epimeric vitamin Danalyte and a second antibody that is antibody 8F10 and then with thechemibead reagent. The chemibeads bind to the fraction of the monoclonalantibody binding sites that is not occupied by non-epimeric vitamin Danalyte from the sample. Antibody 8F10 binds to 3-epi-25-hydroxyvitaminD₃ that is present in the sample and that interferes in thedetermination by cross-reaction with the biotinylated antibody.Subsequently, streptavidin coupled sensitizer beads comprising aphotosensitizer (sensibeads) are added to the reaction mixture. Thisleads to the formation of chemibead/sensibead pairs whose concentrationis inversely related to a concentration of non-epimeric vitamin Danalyte in the sample. Upon illumination at 680 nm, the sensitizer beadsgenerate singlet oxygen which diffuses into the chemibeads, which arepaired with sensibeads, reacts with the olefinic dye in thechemiluminescent and triggers a chemiluminescent signal at approximately612 nm which is inversely related to the non-epimeric vitamin D analyteconcentration.

Examples of Assays for Vitamin D Analytes Employing a Labeled Compoundin Accordance with the Principles Described Herein

Assays for vitamin D analytes may be carried out using a compound of theFormula I wherein Z is a poly(amino acid) label, or a non-poly(aminoacid) label or a support.

In an example, by way of illustration and not limitation, of an assayfor detection of vitamin D analyte, an ACMIA assay format is employed.Chrome particles, which are coated with a vitamin D analog that is acompound of the Formula I wherein Z is a non-poly(amino acid) label thatis the chrome particle (chrome particle reagent), are employed as afirst component. A second component is an antibody for vitamin Danalyte. This antibody, crosslinked to a reporter enzyme (for example,β-galactosidase) to form an antibody-enzyme conjugate, is added to areaction vessel in an excess amount, i.e., an amount greater than thatrequired to bind all of the vitamin D analyte that might be present in asample. A sample, which is previously subjected to treatment with areleasing agent, is treated with an antibody for vitamin D, which bindsto vitamin D analyte in the sample. The antibody-enzyme conjugate ismixed with sample in the medium to allow the vitamin D analyte to bindto the antibody. Next, the chrome particle reagent is added to bind upany excess antibody-enzyme conjugate. Then, a magnet is applied, whichpulls all of the chrome particles and excess antibody-enzyme out of thesuspension, and the supernatant is transferred to a final reactioncontainer. The substrate of the reporter enzyme is added to the finalreaction container, and the enzyme activity is measuredspectrophotometrically as a change in absorbance over time. The amountof this signal is related to the amount of vitamin D analyte in thesample.

In the above example, an antibody in accordance with the principlesdescribed herein may be employed to block binding of3-epi-25-hydroxyvitamin D₃, which may be present in the sample, to theantibody for the vitamin D analyte. The anti-3-epi-25-hydroxyvitamin D₃antibody may be, an antibody raised against an immunogen that is acompound of Formula I wherein Z is an immunogenic carrier.

Examination Step

In a next step of an assay method, the medium is examined for thepresence of a complex comprising the vitamin D analyte and antibody forthe vitamin D analyte and/or a complex comprising a vitamin D analog andantibody for vitamin D. The presence and/or amount of one or both of thecomplexes indicates the presence and/or amount of the vitamin D analytein the sample.

The phrase “measuring the amount of a vitamin D analyte” refers to thequantitative, semiquantitative and qualitative determination of vitaminD. Methods that are quantitative, semiquantitative and qualitative, aswell as all other methods for determining the vitamin D analyte, areconsidered to be methods of measuring the amount of the vitamin Danalyte. For example, a method, which merely detects the presence orabsence of the vitamin D analyte in a sample suspected of containing thevitamin D analyte, is considered to be included within the scope of thepresent invention. The terms “detecting” and “determining,” as well asother common synonyms for measuring, are contemplated within the scopeof the present invention.

In many embodiments the examination of the medium involves detection ofa signal from the medium. The presence and/or amount of the signal isrelated to the presence and/or amount of the vitamin D analyte in thesample. The particular mode of detection depends on the nature of thesignal producing system. As discussed above, there are numerous methodsby which a label of a signal producing signal can produce a signaldetectable by external means. Activation of a signal producing systemdepends on the nature of the signal producing system members.

Temperatures during measurements generally range from about 10° C. toabout 70° C. or from about 20° C. to about 45° C., or about 20° C. toabout 25° C., for example. In one approach standard curves are formedusing known concentrations of vitamin D analyte. Calibrators and othercontrols may also be used.

Luminescence or light produced from any label can be measured visually,photographically, actinometrically, spectrophotometrically, such as byusing a photomultiplier or a photodiode, or by any other convenientmeans to determine the amount thereof, which is related to the amount ofvitamin D analyte in the medium. The examination for presence and/oramount of the signal also includes the detection of the signal, which isgenerally merely a step in which the signal is read. The signal isnormally read using an instrument, the nature of which depends on thenature of the signal. The instrument may be, but is not limited to, aspectrophotometer, fluorometer, absorption spectrometer, luminometer,and chemiluminometer, for example.

Kits Comprising Reagents for Conducting Assays

Kits comprising reagents for conducting assays can be formulated basedon the nature of a particular assay. In some examples in accordance withthe principles described herein a kit can comprise a binding partnersuch as, for example, an antibody raised against an immunogen that is acompound of the Formula I wherein Z is an immunogenic carrier. In someexamples in accordance with the principles described herein, a kit cancomprise a reagent that is a compound of the Formula I wherein Z is apoly(amino acid) label moiety or a non-poly(amino acid) label moietyincluding a support. A kit may also include other reagents forconducting a particular assay for a vitamin D analyte. In someembodiments a kit comprises in packaged combination a biotin-bindingpartner such as, for example, avidin or streptavidin, associated with aparticle, biotinylated compound of Formula I and a labeled antibody forthe vitamin D analyte. The kit may further include other reagents forperforming the assay, the nature of which depend upon the particularassay format.

The reagents may each be in separate containers or various reagents canbe combined in one or more containers depending on the cross-reactivityand stability of the reagents. The kit can further include otherseparately packaged reagents for conducting an assay such as additionalspecific binding pair members, signal producing system members, andancillary reagents, for example.

The relative amounts of the various reagents in the kits can be variedwidely to provide for concentrations of the reagents that substantiallyoptimize the reactions that need to occur during the present methods andfurther to optimize substantially the sensitivity of an assay. Underappropriate circumstances one or more of the reagents in the kit can beprovided as a dry powder, usually lyophilized, including excipients,which on dissolution will provide for a reagent solution having theappropriate concentrations for performing a method or assay using acompound reagent in accordance with the principles described herein. Thekit can further include a written description of a method utilizingreagents that include a compound reagent in accordance with theprinciples described herein.

Pharmaceutical Compositions Containing Compounds in Accordance with thePrinciples Described Herein

Examples of compounds and antibodies in accordance with the principlesdescribed herein can have therapeutic activity. These compounds may beadministered in therapeutically effective amount, which is an amount toprovide treatment of a particular disease state associated with vitaminD. Administration of the examples of compounds in accordance with theprinciples described herein can be by means of any of the accepted modesof administration for agents that serve similar utilities. The compoundscan be used both prophylactically and therapeutically.

The amount of compound administered is dependent on one or more of thesubject and disease state being treated, the severity of the affliction,the manner and schedule of administration (e.g., oral administration)and the judgment of the prescribing physician.

In employing the compounds of this invention for treatment of the aboveconditions, any pharmaceutically acceptable mode of administration canbe used. The compounds in accordance with the principles describedherein can be administered either alone or in combination with otherpharmaceutically acceptable excipients, including, but not limited to,solid, semi-solid, liquid or aerosol dosage forms, such as, for example,tablets, capsules, powders, liquids, injectables, suspensions,suppositories, and aerosols, for example. The compounds can also beadministered in sustained or controlled release dosage forms, includingdepot injections, osmotic pumps, pills, and transdermal (includingelectrotransport) patches, for example, for the prolonged administrationof the compound at a predetermined rate, preferably in unit dosage formssuitable for single administration of precise dosages. Thepharmaceutical compositions include one or more conventionalpharmaceutical carriers or excipients and a compound in accordance withthe principles described herein. In addition, these compositions mayinclude other medicinal agents, pharmaceutical agents, carriers, andadjuvants, for example.

Depending on the intended mode of administration, a pharmaceuticallyacceptable composition will contain about 0.1% to about 90%, by weightof a compound in accordance with the principles described herein, theremainder being suitable pharmaceutical excipients and carriers, forexample.

One mode of administration for the conditions detailed above is oral,using a convenient daily dosage regimen, which can be adjusted accordingto the degree of affliction. For such oral administration, apharmaceutically acceptable composition is formed by the incorporationof any of the normally employed excipients, such as, for example,mannitol, lactose, starch, povidone, magnesium stearate, sodiumsaccharine, talcum, cellulose, croscarmellose sodium, glucose, gelatin,sucrose, and magnesium carbonate, for example, and combinations of twoor more thereof. Such compositions may be solutions, suspensions,tablets, dispersible tablets, pills, capsules, powders, or sustainedrelease formulations, for example.

In one approach by way of illustration and not limitation, a compositionmay be in the form of a pill or tablet and may contain, along with acompound in accordance with the principles described herein, a diluentsuch as lactose, sucrose, or dicalcium phosphate, for example, orcombination of two or more thereof; a lubricant such as magnesiumstearate, for example; a disintegrant such as croscarmellose sodium, forexample; and a binder such as starch, gum acacia, polyvinylpyrrolidine,gelatin, cellulose and derivatives thereof, for example.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients include, butare not limited to, water, saline, dextrose, glycerol and ethanol, forexample. In addition, the pharmaceutical compositions to be administeredmay also contain minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and solubilityenhancers, for example, such as, for example, sodium acetate,polyoxyethylene, sorbitan monolaurate, triethanolamine oleate, andcyclodextrins.

In one example in accordance with the principles described herein, apharmaceutical composition comprises a compound of the formula:

wherein W is O or N—O—(CH₂)_(n″)C(O)—Z″,

-   -   n″ is an integer from 1 to 10,    -   R^(11″) is H, alkyl, or acyl, and    -   Z″ is OR⁸ wherein R⁸ is H, alkyl, or NR⁹R¹⁰ wherein R⁹ and R¹⁰        are independently H or alkyl.

The phrase “at least” as used herein means that the number of specifieditems may be equal to or greater than the number recited. The phrase“about” as used herein means that the number recited may differ by plusor minus 10%; for example, “about 5” means a range of 4.5 to 5.5.

The following discussion is directed to specific examples in accordancewith the principles described herein by way of illustration and notlimitation; the specific examples are not intended to limit the scope ofthe present disclosure and the appended claims. Numerous modificationsand alternative compositions, methods, and systems may be devisedwithout departing from the spirit and scope of the present disclosure.

EXAMPLES

Unless otherwise indicated, materials in the experiments below may bepurchased from the Sigma-Aldrich Chemical Corporation (St. Louis Mo.),Fluka Chemical Corporation (Milwaukee Wis.), or Steraloids Inc. (WiltonN.H.). Parts and percentages disclosed herein are by weight to volumeunless otherwise indicated.

Definitions

-   -   mg=milligram    -   g=gram(s)    -   ng=nanogram(s)    -   mL=milliliter(s)    -   μL=microliter(s)    -   μmol=micromolar    -   ° C.=degrees Centigrade    -   min=minute(s)    -   sec=second(s)    -   hr=hour(s)    -   w/v=weight to volume    -   TLC=thin layer chromatography    -   HPLC=high performance liquid chromatography    -   EDA=ethylenediamine    -   EtOAc=ethyl acetate    -   DMF=dimethylformamide    -   DMSO=dimethylsulfoxide    -   MeOP=1-methoxy-2-propanol    -   MES=2-(N-morpholino)ethanesulfonic acid    -   CMO=carboxymethoxyoxime    -   TMB=tetramethyl benzidine    -   SNHS=sulfo-N-hydroxysuccinimide    -   High pH Wash Buffer=5.5 mM Na₃PO₄+4.4 mM Na₂CO₃, pH 11    -   Hapten Wash Buffer=50 mM HEPES, 300 mM NaCl, 1 mM EDTA, 0.1%        TRITON® X405, 0.15% PROCLIN® preservative, and 1 mg/ml neomycin    -   DI=deionized    -   ELISA=enzyme-linked immunosorbent assay    -   UPA=Ultra Particle Analyzer    -   LOCI=luminescent oxygen channeling immunoassay    -   BSA=bovine serum albumin    -   BGG=bovine gamma globulin    -   mIgG=mouse immunoglobulin    -   MS=mass spectrometry

Example 1

Preparation of Compounds of FIG. 2 where R¹¹ is Acetyl

5-Androsten-3α-ol-17-one acetate (VIII) (100 mg) was reacted withethylene glycol (0.245 ml) and p-toluenesulfonic acid monohydrate (4 mg)in benzene under reflux overnight to give 5-androsten-3α-ol-17-oneacetate ethylene ketal (IX) (112 mg). The ethylene ketal IX (112 mg) wasbrominated with N-bromosuccinimide (69 mg)/azoisobutyronitrile (3.3 mg)in hexane (13 ml) under reflux for 30 minutes to give compound X,followed by dehydrobromination with tetrabutylammonium fluoride (IM inTHF, 1.6 ml) in THF (7.3 ml) at room temperature for 2 hours to giveandrosta-5,7-dien-3α-ol-17-one acetate ethylene ketal (XI) (55 mg).Androsta-5,7-dien-3α-ol-17-one acetate ethylene ketal (XI) (55 mg) wasreacted with IN sodium hydroxide (2 mL) in methanol (10 ml) at roomtemperature for about 5 hours to give androsta-5,7-dien-3α-ol-17-oneethylene ketal (XII) (48 mg). Androsta-5,7-dien-3α-ol-17-one ethyleneketal (XII) (48 mg) was reacted with p-toluenesulfonic acid monohydrate(35 mg) in a mixture of acetone (5 mL) and water (0.2 mL) at roomtemperature overnight to give androsta-5,7-dien-3α-ol-17-one (XIII)(43.4 mg). Androsta-5,7-dien-3α-ol-17-one (XIII) (43.4 mg) wasirradiated under 450 w mercury lamp with a VYCOR® filter (Ace GlassIncorporated, Vineland, N.J.) in ether (1100 ml) at −20 to 0° C. for 5minutes to give pre-HMCHEMOIO, which was refluxed in ethanol (15 ml) for3 hours to produce HMCHEMOIO (VII) (13.5 mg).

The HMCHEMOIO (VII) (13.5 mg) from above was reacted withO-(carboxymethyl)hydroxylamine hemihydrochloride (12 mg) and sodiumacetate (24 mg) in 1 ml methanol for overnight at room temperature togive HMCHEMOIO-CMO (13.2 mg) (compound of the Formula XX in FIG. 3 wheren=1).

Cationized BSA was prepared as follows: Triethylenetetraamine (0.5 mL)(compound of the Formula XXI in FIG. 3 where each r=1 and s=1) was addedto 4.5 ml 50 mM MES buffer pH6, and the pH was adjusted to 6 followed byaddition of 20 mg BSA. The combination was mixed to completely dissolvethe components. EDAC (5 mg) was added to the above solution each hourfor 5 hours. The solution was washed in a 100 mL AMICON® cell (AmiconInc., Beverly Mass.) with 10×10 mL washing buffer (10 mM phosphate+300nM NaCl) at pH7. Then, 100 mg TWEEN®20 was added to a 50-mL round bottomflask. Washing buffer (50 mL) was added and the mixture was stirred for10 min to disperse Tween 20. The cationized BSA (compound of the FormulaXXIa in FIG. 3 where each r=1 and s=2) was stored in washing buffer with0.2% TWEEN® 20 at about 5 mg/mL.

For preparation of HMCHEMOIO-CMO BSA (compound of the Formula IIa inFIG. 3), EDAC (25 mg) and 10 mg NHS were placed into a 10 ml flask with13.2 mg HMCHEMOIO-CMO (XX) prepared as described above. DMF (0.5 ml) wasadded to the flask. The mixture was stirred for 24 hours at roomtemperature to give activated HMCHEMOIO-CMO. The solution was clear. TLC(1:1 Ethyl Acetate:Methanol) indicated no remaining starting material.

The activated HMCHEMOIO-CMO from above was added drop wise to thecationized BSA solution from above, and the mixture was stirredovernight at room temperature. The mixture was transferred to an AMICON®cell (Amicon Inc., Beverly Mass.), and then washed with 5×10 ml washingbuffer and concentrated (30,000 cut off) to around 4 mL. The mixture wasfurther separated on an SD-25 column (GE Healthcare Bio-Sciences,Pittsburgh Pa.) using washing buffer pH 7.0 as an elutant to give amixture of HMCHEMOIO-CMO BSA compounds (mixture as discussed above withregard to compounds of the Formula IIa in FIG. 3).

Example 2

Preparation of Antibodies

AJ strain of mice (females, at least eight weeks old) was immunized togenerate monoclonal antibodies. The first immunization was 100 μg ofimmunogen (HMCHEMOIO-CMO BSA) from above in a volume of 100 μl withComplete Fruends' adjuvant (from Sigma-Aldrich, Cat #F5881). Three weekslater, a boost immunization with the same immunogen was given with 100μg in a volume of 100 μL with Incomplete Fruend's adjuvant (fromSigma-Aldrich Cat #F5506). Subsequently, after another 3 weeks, a secondbooster immunization with the same immunogen was given with 100 g in avolume of 100 μL with Incomplete Fruends' Adjuvant. One week followinglast booster immunization, mice were bled and anti-sera were tested inELISA for anti-3-epimer antibodies. Subsequently, a prefusion boost wasgiven on three consecutive days before fusion with the same immunogen(20 μg in a volume of 50 μL in PBS without any adjuvant. On the fourthday, mice were sacrificed and splenectomy was performed. Spleen cellswere removed and fusion was performed by standard methods using anon-secreting murine myeloma cell line designated P3-X63Ag8.653 (ATCCCRL-1580™). Cloning was done by standard methods.

The clones were screened by binding and inhibition ELISA. The followingbinding ELISA immunoassay procedure according to the following protocol.Plates were coated with 3-epimer-conjugated to ovalbumin prepared in amanner similar to that described above for the BSA conjugate at 1 μg/mLin PBS at 50 μL per well. Plate coating was performed for 1 hour or moreat room temperature. The plates were then flicked dry and blocked with200 μL per well of blocking buffer diluent (0.5% Casein solution in PBScontaining 0.05% Tween® 20). Plate blocking was performed for 1 hour orovernight at 2° C.-8° C. The plates were then washed three times andflicked dry. The monoclonal antibody to be screened was then added toeach well as follows: 25 μL of PBS mixed with 25 μL of culturesupernatant transferred from the corresponding well in the fusion growthplate. Incubation was for about 1 hour at room temperature with plateshaking. The plate was washed using a plate washer (BioTek, WinooskiVt.) with plate stacker with the washing buffer being MILLI-Q® water(Millipore Corporation, Billerica, Mass.) containing 0.05% Tween® 20. Anenzyme conjugate (goat anti-mouse IgG coupled to HRP diluted in blockingbuffer diluent to 1:3000 was added at 50 μL per well. Incubation wasperformed for about 1 hour at room temperature with shaking. The platewas then washed and a chromogenic solution (TMB from Moss Substrates,Pasadena Md.) was added at a volume of 100 μL per well for ten minutesat room temperature. Plates were read at 650 nm using an ELISA platereader.

Based on the above screening technique, hybridomas producing suitablemonoclonal antibodies were selected. One such monoclonal antibody wasdesignated antibody 8F10 and is an IgG2a kappa antibody. Another suchmonoclonal antibody was designated antibody 4G8 and is an IgG2a kappaantibody.

Additionally, clones were also screened using an inhibition ELISAprocedure according to the following protocol. Plates were coated with3-epimer-conjugated to ovalbumin at 1 μg/mL in phosphate buffered salineat 50 μL per well. Plate coating was performed for 1 hour or more atroom temperature or overnight at about 2° C. to 8° C. The plates werethen flicked dry and blocked with 200 μL per well of blocking bufferdiluent (0.5% Casein solution in PBS containing 0.05% TWEEN® 20). Plateblocking was performed by incubation for 30 min or more at roomtemperature with plate shaking. The plates were washed. The monoclonalantibody to be screened was then added to each well along with free3-epimer as follows: 25 μL per well culture supernatant transferred fromthe corresponding well in the fusion growth plate and added 25 μL of 2g/mL of 3-epimer-25OH Vitamin D₃. Incubation was for about 1 hour atroom temperature with plate shaking. The plate was washed using a platewasher (BioTek, Winooski Vt.) with plate stacker with the washing bufferbeing MILLI-Q® water (Millipore Corporation, Billerica, Mass.)containing 0.05% TWEEN® 20. An enzyme conjugate (goat anti-mouse IgGcoupled to HRP diluted in blocking buffer diluent to 1:3000 was added at50 μL per well. Incubation was performed for about 1 hour at roomtemperature with shaking. The plate was then washed and a chromogenicsolution (TMB from Moss Substrates, Pasadena Md.) was added at a volumeof 100 μL per well. If a desired antibody was present in the hybridomasupernatant, then a decrease in optical density was observed compared tothe well containing no free 3-epimer. 25OH Vitamin D₃ was used in placeof free 3-epimer-25 OH Vitamin D₃ as a control to monitor thespecificity of the antibodies. Antibodies binding to 3-epimer and notbinding to 25OH vitamin D3 were selected.

Polyclonal antibody generation: Rabbits were immunized with 500 μg/doseof HMCHEMOIO-CMO BSA prepared as described above. One primary and fivebooster immunizations two weeks apart were performed and two test bleedsand one production bleed were collected and anti-sera was tested inELISA as described above.

Example 3 Immunoassay for 25OH Vitamin D

Immunoassay Procedure

The 25OH Vitamin D (25(OH)D) immunoassay format employed was ahomogeneous competitive chemiluminescent immunoassay based on LOCI®assay technology. The assay was performed on the Siemens Dimension® EXLautomated integrated clinical chemistry system (Siemens HealthcareDiagnostics Inc., Deerfield, Ill.). The assay measured the totalconcentration of 25(OH)D₂ and/or 25(OH)D₃ in serum and plasma samples.The LOCI® assay reagents included a releasing reagent, two syntheticbead reagents and a biotinylated monoclonal anti-25OH vitamin D antibodyreagent. The first bead reagent (designated “Sensibeads”) was coatedwith streptavidin and contained a photosensitive dye. The second beadreagent (designated “Chemibeads”) was coated with a 25(OH)D₃ analog andcontained chemiluminescent dye. Sample was incubated with the releasingreagent to release 25(OH)D molecules including 3-epimeric compounds fromvitamin D binding proteins. The reaction mixture was then incubated withbiotinylated antibody to form a 25(OH)D-biotinylated antibody complex.Because the biotinylated antibody (a sheep monoclonal antibody) crossreacted with 3-epi-25(OH)D, 100 μg/mL of anti-3-epimer-VD antibody8F10.1 was added to minimize assay signal coming from the 3-epimervitamin D compounds. Chemibeads were added to bind excess freebiotinylated antibody. Sensibeads were then added to bind to the biotinportion of the biotinylated antibody. Aggregates ofChemibead-analog/antibody-biotin/streptavidin-Sensibeads were formed asa result. Illumination of the reaction mixture with light at 680 nmgenerates singlet oxygen from Sensibeads, which diffused into theChemibeads and triggered a chemiluminescent reaction. The resultingchemiluminescent signal was measured at 612 nm and is inverselyproportional to the concentration of total 25(OH)D in the sample.

Preparation of Reagents for Immunoassay

Synthesis of 25(OH)D₃ Chemibeads—

The 25(OH)D₃ Chemibeads were synthesized by coupling EPRM-EDA with25(OH)D₃ carbamate. The materials employed were EPRM-EDA Beads,1-Ethyl-3-(3-Dimethyl Aminopropyl)Carbodiimide (EDAC) FlukaSulfo-N-Hydroxysuccinimide (SNHS), 25(OH)D₃-3-Carbamate, GAFAC®surfactant solution 16%, Anhydrous DMSO, 50 mM MES pH6 Buffer containing10% MeOP and 1% GAFAC® surfactant.

Preparation of EPRM-EDA Beads—

EPRM beads (2000 mg, 20.0 mL) are added to a 40-mL vial. The EPRM beadsare prepared by a procedure similar to that described in U.S. Pat. No.7,179,660 and the chemiluminescent compound is 2-(4-(N,N,di-tetradecyl)-anilino-3-phenyl thioxene with europium chelate. EDA (800mg, 890 μL) is combined with 10 mL MES pH 6 buffer (the “Buffer”) andabout 4.2 mL 6N HCl. The pH of the mixture is, or is adjusted to be,about 6.9. The EDA solution is added to the EPRM beads with vortexingand the mixture is rocked at room temperature for 15 minutes. Sodiumcyanoborohydride (400 mg) is combined in a 15 mL vial with 10 mL DIwater and the combination is added to the bead mixture from above. Themixture is shaken at 37° C. for 18-20 hours. The beads are transferredto six 40 mL centrifuge tubes. MES buffer is added to bring the volumeto 35 mL and the mixture is centrifuged at 19,000 rpm for 30 min. Thesupernatant is decanted and the beads are re-suspended in 2 mL of theBuffer with a stir-rod and additional Buffer is added to 35 mL. Themixture is sonicated at 18 Watts power for 30 sec, using ice to keep themixture cold. The wash/sonication step is performed 4 times to removeall activation chemical. After the last MES Buffer centrifugation, 2 mLof the Buffer containing 5% MeOP and 0.1% Tween® 20 (the “secondBuffer”) is added to the tubes for the re-suspension step. Additionalsecond Buffer is added to 35 mL before sonication. The bead suspensionis centrifuged at 19,000 rpm for 30 min. The supernatant is discarded.The final sonication used 12 mL of the second Buffer in each tube togive a 25 mg/mL dilution. Particle size is 277 nm as determined on a UPAinstrument.

The EPRM chemibead is prepared in a manner similar to the methoddescribed in U.S. Pat. No. 6,153,442 and U.S. Patent ApplicationPublication No. 20050118727A, the relevant disclosures of which areincorporated herein by reference. The EPRM chemibead comprises anaminodextran inner layer and a dextran aldehyde outer layer having freealdehyde functionalities. See, for example, U.S. Pat. Nos. 5,929,049,7,179,660 and 7,172,906, the relevant disclosures of which areincorporated herein by reference. The reaction is carried out at atemperature of about 0 to about 40° C. for a period of about 16 to about64 hours at a pH of about 5.5 to about 7.0, or about 6, in a bufferedaqueous medium employing a suitable buffer such as, for example, MES.The reaction is quenched by addition of a suitable quenching agent suchas, for example, carboxymethoxyamine hemihydrochloride (CMO), andsubsequent washing of the particles.

Aldehyde groups on the outer dextran aldehyde layer are reacted withethylene diamine under reductive amination conditions to form reagentEPRM-EDA having pendant moieties comprising an ethylene chain and aterminal amine group. The reductive amination conditions include the useof a reducing agent such as, for example, a metal hydride. The reactionis carried out in an aqueous medium at a temperature during the reactionof about 20° C. to about 100° C. for a period of about 1 hour to about48 hours.

Synthesis of 25(OH)D₃-3-carbamate (25(OH)D₃-3-carbamate)—

A mixture of 22 mg (55 μmol) 25(OH)D₃ purchased from ChemReagents.com,Sugarland Tex., 100 mg (420 μmol) disuccinimidyl carbonate (DSC), 100 μLtriethylamine in 1 mL anhydrous acetonitrile in a 5-mL flask (coveredwith foil) was stirred at room temperature for 18 hr under nitrogen toprepare activated 25(OH)D₃. TLC (EtOAc:Hexane=2:1) showed no startingmaterial left. A suspension was prepared by adding 150 mg ofcarboxymethoxylamine hemihydrochloride (CMO), 0.3 ml triethylamine and 1ml DMF to a 10 ml flask. A solution containing activated 25(OH)D₃ wasadded dropwise to the CMO suspension with stirring, which was continuedfor another 18 hr. Vacuum was applied to remove the solvents as much aspossible (keeping the heating bath temperature below 50° C.). EtOAc (25ml) was added to the residue, which was washed three times with 2 mlbrine. The organic phase was dried with anhydrous Na₂SO₄ and wasfiltered; solvent was removed using a rotavap. Crude product (42 mg) wasobtained after drying and was purified by HPLC. Pure product (24 mg) wasobtained after drying under high vacuum. The product was dissolved into1.2 ml anhydrous DMSO. Aliquots were transferred into vials, which werekept at −70° C.

Coupling EPRM-EDA with the Hapten 25(OH)D₃-Carbamate—

1.2 mg hapten was added to a 2-mL vial. 11.2 mg EDAC and 15.5 mg SNHSplus 3.73 mL dry DMSO was added to a 5-mL vial. EDAC/SNHS solution wasrotated to dissolve contents. 1.14 mL EDAC/SNHS solution was added tothe vial containing hapten. The mixture was rotated for 22 hours.EPRM-EDA (200 mg) was washed once with High pH Wash Buffer and then withMES pH6 buffer. To a 5-mL vial was added 1.08 mL (100 mg) of wash bufferfollowed by 143 mL 1.6% GAFAC® surfactant. To a small test tube wasadded 256 dry DMSO followed by 49 μL EDAC/SNHS/hapten. The DMSO/haptensolution was added drop-wise to the bead mixture (subjected to vortexingduring addition). The bead/hapten mixture was rotated overnight at roomtemperature.

The bead/hapten mixture was transferred to a 50-mL centrifuge tube anddiluted to 35 mL with 10% 1-methoxy-2-propanol/1% GAFAC® surfactant/MESpH6 buffer. The tube was centrifuged at 18,500 rpm at 10° C. for 30 min.The supernatant was discarded and replaced by 1 mL of the same buffer.The pellet was re-suspended with a stir-rod. The vial was filled to 35mL with the same buffer. The tube was sonicated at 18-21 Watts for 1minute using ice to keep the tube cold. The centrifugation/washing wasrepeated six times. Following the sixth wash, the buffer was switched toHapten Wash Buffer pH7.2 and two more washings performed. Following thelast wash and re-suspension with 1 mL Hapten wash buffer, 4 mL HaptenWash Buffer was added. The bead mixture was sonicated at 50% power in acup sonicator. Particle size was measured by UPA as 298 nm. Percentsolids assay was performed and the bead mixture was diluted to 10 mg/mL.This Chemibead Reagent was formulated in 50 mM MES buffer.

Biotinvlation of Anti-25(OH)D Antibody—

NHS-PEO₄-biotin (Pierce Chemical Company, Rockford Ill.) was coupledwith the anti-25(OH)D antibody (a sheep monoclonal antibody fromBioventix, Farnham, Surrey, UK). 3 mg of the anti-25(OH)D antibody wasbuffer-exchanged twice with 10 ml each of Antibody Dialysis Buffer (10mM NaH₂PO₄ pH 7.0/300 mM NaCl) in 10 mL Amicon and then concentrated to3.0 mg/mL. 1 mg of NHS-PEO4-biotin was dissolved in 100 μL AntibodyDialysis Buffer to make 10 mg/mL of the biotin reagent solution, whichwas added (35 μL) to the anti-25(OH)D antibody solution. The reactionmixture was rocked at room temperature overnight. The mixture was washedthree times with 10 mL each of Antibody Dialysis Buffer in 10 mL Amicon,and then concentrated to about 1 mL. The concentration was measured onUV A280. The biotinylated anti-25(OH)D antibody reagent was formulatedin an aqueous buffer containing 25 mM citric acid buffer, 300 mM NaCl, 1mM EDTA, blocking proteins and preservatives, pH 5.0.

Sensibeads—

Streptavidin-sensitizer bead was prepared using a method analogous tothat described in U.S. Pat. Nos. 6,153,442, 7,022,529, 7,229,842 andU.S. Patent Application Publication No. 20050118727A. Thephotosensitizer was bis-(trihexyl)-silicon-t-butyl-phthalocyanine. Theconcentration of Sensibead reagent was 200 μg/mL in HEPES buffer, pH 8.0containing 150 mM NaCl.

Releasing Reagent—

Sodium salicylate in 5 mM HEPES buffer.

Results of Immunoassay

The effect of adding anti-3-epimer-VD antibody 8F10.1 (anti-3-epimer VDAb) on the 3-epimer cross-reactivity for the assay is shown in Table 1.Ten patient sera containing 25(OH)D₂ or 25(OH)D₃ compounds were spikedwith 100 ng/mL of 3-epimer vitamin D₃ compound purchased from Sigma(Stock No. 751324). The 3-epimer cross-reactivity was calculated by thedifference between 25(OH)D₂ or 25(OH)D₃ values with and without thespiked 3-epimer compound divided by the amount of added 3-epimercompound. With addition of 100 μg/mL of the anti-3-epimer-VD antibody8F10.1, the average 3-epimer cross-reactivity was reduced from 13.7% toless than 2%.

TABLE 1 No anti-3-epimer Anti-3-epimer VD Ab Added VD Ab Added Sample3-epimer Cross- Cross- ID D3 Spiked ng/mL Reactivity ng/mL Reactivity 1No 30.2 30.7 Yes 48.0 17.9% 33.0 2.2% 2 No 22.5 23.2 Yes 40.5 17.9% 25.62.4% 3 No 19.8 20.7 Yes 31.6 11.9% 22.5 1.8% 4 No 40.4 40.5 Yes 53.012.6% 41.4 1.0% 5 No 46.6 45.6 Yes 62.7 16.1% 49.2 3.5% 6 No 47.3 48.5Yes 57.5 10.2% 46.4 −2.0% 7 No 53.1 55.4 Yes 62.2 9.1% 55.4 0.0% 8 No39.5 40.8 Yes 50.0 10.5% 43.8 3.0% 9 No 47.4 48.6 Yes 62.8 15.3% 50.82.2% 10 No 23.6 24.8 Yes 39.1 15.5% 28.6 3.8% Avg. Cross-reactivity13.7% 1.8%

FIG. 9 illustrates a comparison of standard curves generated usingreagents with and without added anti-3-epimer-VD antibody 8F10.1(3-epimer Ab). The complete overlap of the two curves shows that theanti-3-epimer-VD antibody 8F10.1 does not affect the measurement of25(OH)D₃ (25OH Vit D3 in FIG. 9), indicating the antibody is specific tothe 3-epimer compound and has no observable cross-reactivity with the25(OH)D₃.

Example 4 Immunoassay for 3-epi-25OH Vitamin D

Immunoassay Procedure

The measurement of 3-epi-25OH Vitamin D (3-epi-25(OH)D) employed ahomogeneous competitive chemiluminescent immunoassay based on LOCI®assay technology similar to that described above in Example 3. The assaywas performed on the Siemens Dimension® EXL automated integratedclinical chemistry system (Siemens Healthcare Diagnostics Inc. The LOCI®assay reagents utilized for the measurement of 3-epi-25(OH)D included areleasing reagent, two synthetic bead reagents and a biotinylatedmonoclonal anti-3-epimer antibody reagent. The first bead reagent(Sensibeads) was coated with streptavidin and contained photosensitivedye. The second bead reagent (Chemibeads) was coated with a 3-epimeranalog (compound of the Formula IIa above wherein Z′ is a non-poly(aminoacid) label, namely, EPRM-EDA chemibead) prepared in a manner similar tothe described above for Example 1. Sample was incubated with thereleasing reagent to release 25(OH)D molecules including 3-epimericcompounds from vitamin D binding proteins. The reaction mixture was thenincubated with biotinylated antibody to form a3-epi-25(OH)D/biotinylated antibody complex. Chemibeads were added toscavenge the excess free biotinylated antibody. Sensibeads are thenadded and bind to the biotin portion of the biotinylated antibody.Aggregates of Chemibead-analog/antibody-biotin/streptavidin-Sensibeadswere formed as a result. Illumination of the reaction mixture by lightat 680 nm generated singlet oxygen from the Sensibeads, which diffusedinto the Chemibeads and triggered a chemiluminescent reaction. Theresulting chemiluminescent signal is measured at 612 nm and is inverselyproportional to the concentration of total 3-epi-25(OH)D in the sample.

Preparation of Reagents for Immunoassay

Synthesis of 3-Epimer Analog Chemibeads—

Chemibeads for use in this assay for the detection of 3-epi-25(OH)D wereprepared in a manner similar to that described above in Example 3 forSynthesis of 25(OH)D3 Chemibeads with the hapten being a compound of theFormula IIa where Z′ is the chemibead.

Chemibead Reagent—3-epimer analog Chemibeads in 50 nM MES buffer.

Biotinylation of Anti-3-Epimer-VD Antibody 8F10.1—

Biotinylation of the anti-3-epimer-VD antibody 8F10.1 was carried out ina manner similar to that described above in Example 3 for theBiotinylation of anti-25(OH)D antibody.

Biotinylated Antibody Reagent—

Biotinylated anti-3-epimer-VD antibody 8F10.1 stated above in 25 mMcitric buffer.

Sensibead Reagent—

Sensibead Reagent was the same as in Example 3.

Results of Immunoassay

FIG. 10 illustrates a standard curve for the immunoassay for thedetermination of 3-epi-25(OH)D in samples using anti-3-epimer-VDantibody 8F10.1. Chemiluminescent Kilocounts represents thechemiluminescent signal measured as described above.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

It should be understood that the above-described examples are merelyillustrative of some of the many specific examples that represent theprinciples described herein. Clearly, those skilled in the art canreadily devise numerous other arrangements without departing from thescope as defined by the following claims.

What is claimed is:
 1. A composition comprising one or more of acompound of the formula:NHR^(1′)—(CH₂)_(r′)—NR^(1″)—((CH₂)_(r′)—NR^(1′″))_(s′)—(CH₂)_(r′)—NR^(7′)—Z′wherein R^(1′), R^(1″) or R^(1′″) are each independently selected from

 and H, wherein at least one of R^(1′), R^(1″)or R^(1′″) is not H, n isan integer from 1 to 10, r′ is independently an integer from 1 to 10, s′is an integer from 1 to 10, R^(7′)is H or alkyl, R^(11′) is H, alkyl, oracyl, and Z′ is a poly(amino acid) immunogenic carrier, a non-poly(aminoacid) immunogenic carrier, a poly(amino acid) label moiety, anon-poly(amino acid) label moiety, a non-label poly(amino acid) moiety,or a non-immunogenic carrier poly(amino acid) moiety.
 2. The compositionaccording to claim 1 wherein s′ of the compound is
 1. 3. The compositionaccording to claim 1 wherein R^(7′) of the compound is H.
 4. Thecomposition according to claim 1 wherein r′ of the compound is
 2. 5. Thecomposition according to claim 1 wherein R^(1′) of the compound is

and R^(1″)and R^(1′″) of the compound are H.
 6. The compositionaccording to claim 1 wherein R^(1′) and R^(1″) of the compound are

and R^(1′″) of the compound is H.
 7. The composition according to claim1 wherein R^(1′), R^(1″) and R^(1′″) of the compound are


8. The composition according to claim 1 wherein Z′ of the compound is apoly(amino acid) immunogenic carrier selected from the group consistingof bovine serum albumin, bovine gamma globulin, keyhole limpethemocyanin, and egg ovalbumin.
 9. The composition according to claim 1wherein the poly(amino acid) label of the compound is an enzyme.
 10. Thecomposition according to claim 1 wherein the non-poly (amino acid) labelof the compound is selected from the group consisting of (a)polynucleotides coding for a catalyst, (b) promoters, (c) dyes, (d)fluorescent molecules, (e) chemiluminescent molecules, (f)coenzymes, (g)enzyme substrates, (h) radioactive groups, (i) small organic molecules,and (j) amplifiable polynucleotide sequences.
 11. The composition ofclaim 1 wherein the non-poly(amino acid) label moiety is associated witha support.
 12. The composition of claim 11 wherein the support is aparticle.